3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

ABSTRACT

The present invention relates to the compound of the formula: 
                         
or pharmaceutically acceptable salts thereof, as well as compositions containing the same, processes for the preparation of the same, and therapeutic methods of use therefore in promoting hydration of mucosal surfaces and the treatment of diseases including chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis, acute and chronic bronchitis, cystic fibrosis, emphysema, and pneumonia.

PRIORITY OF INVENTION

This application is a continuation of and claims priority under 35U.S.C. § 120 to U.S. patent application U.S. Ser. No. 14/132,194, filedon Dec. 18, 2013, now issued U.S. Pat. No. 9,586,910, which is adivisional of and claims priority under 35 U.S.C. § 120 to U.S. patentapplication U.S. Ser. No. 13/533,911, filed on Jun. 26, 2012, now issuedU.S. Pat. No. 8,669,262, which claims priority under 35 U.S.C. § 119(e)U.S. Provisional Patent Application, U.S. Ser. No. 61/635,754, filedApr. 19, 2012, and U.S. Provisional Patent Application, U.S. Ser. No.61/501,687, filed Jun. 27, 2011; the entire contents of each of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to novel compounds, particularly including3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl) butyl)carbamimidoyl)pyrazine-2-carboxamide and itspharmaceutically acceptable salt forms, useful as sodium channelblockers, compositions containing the same, therapeutic methods and usesfor the same and processes for preparing the same.

BACKGROUND OF THE INVENTION

The mucosal surfaces at the interface between the environment and thebody have evolved a number of “innate defense”, i.e., protectivemechanisms. A principal form of such innate defense is to cleanse thesesurfaces with liquid. Typically, the quantity of the liquid layer on amucosal surface reflects the balance between epithelial liquidsecretion, often reflecting anion (Cl⁻ and/or HCO₃ ⁻) secretion coupledwith water (and a cation counter-on), and epithelial liquid absorption,often reflecting Na⁺ absorption, coupled with water and counter anion(Cl⁻ and/or HCO₃ ⁻). Many diseases of mucosal surfaces are caused by toolittle protective liquid on those mucosal surfaces created by animbalance between secretion (too little) and absorption (relatively toomuch). The defective salt transport processes that characterize thesemucosal dysfunctions reside in the epithelial layer of the mucosalsurface.

One approach to replenish the protective liquid layer on mucosalsurfaces is to “re-balance” the system by blocking Na⁺ channel andliquid absorption. The epithelial protein that mediates therate-limiting step of Na⁺ and liquid absorption is the epithelial Na⁺channel (ENaC). ENaC is positioned on the apical surface of theepithelium, i.e. the mucosal surface-environmental interface. Ideally,to inhibit ENaC mediated Na⁺ and liquid absorption, an ENaC blocker ofthe amiloride class will be delivered to the mucosal surface andmaintained at this site to achieve maximum therapeutic benefit.

The use of ENaC blockers has been reported for a variety of diseaseswhich are ameliorated by increased mucosal hydration. In particular, theuse of ENaC blockers in the treatment of respiratory diseases such aschronic bronchitis (CB), cystic fibrosis (CF), and COPD, which reflectthe body's failure to clear mucus normally from the lungs and ultimatelyresult in chronic airway infection has been reported. See, Evidence forairway surface dehydration as the initiating event in CF airway disease,R. C. Boucher, Journal of Internal Medicine, Vol. 261, Issue 1, January2007, pages 5-16; and Cystic fibrosis: a disease of vulnerability toairway surface dehydration, R. C. Boucher, Trends in Molecular Medicine,Vol. 13, Issue 6, June 2007, pages 231-240.

Data indicate that the initiating problem in both chronic bronchitis andcystic fibrosis is the failure to clear mucus from airway surfaces. Thefailure to clear mucus reflects an imbalance in the quantities of mucusas airway surface liquid (ASL) on airway surfaces. This imbalanceresults in a relative reduction in ASL which leads to mucusconcentration, reduction in the lubricant activity of the pericilaryliquid (PCL), mucus adherence to the airway surface, and failure toclear mucus via ciliary activity to the mouth. The reduction in mucusclearance leads to chronic bacterial colonization of mucus adherent toairway surfaces. The chronic retention of bacteria, inability of localantimicrobial substances to kill mucus-entrapped bacteria on a chronicbasis, and the consequent chronic inflammatory response to this type ofsurface infection, are manifest in chronic bronchitis and cysticfibrosis.

There is currently a large, unmet medical need for products thatspecifically treat the variety of diseases which are ameliorated byincreased mucosal hydration, including chronic bronchitis, COPD andcystic fibrosis, among others. The current therapies for chronicbronchitis, COPD and cystic fibrosis focus on treating the symptomsand/or the late effects of these diseases. However, none of thesetherapies treat effectively the fundamental problem of the failure toclear mucus from the lung.

R. C. Boucher, in U.S. Pat. No. 6,264,975, describes the use ofpyrazinoylguanidine sodium channel blockers for hydrating mucosalsurfaces typified by the well-known diuretics amiloride, benzamil, andphenamil. However, these compounds are relatively impotent, consideringthe limited mass of drug that can be inhaled to the lung; (2) rapidlyabsorbed, and thereby exhibiting undesirably short half-life on themucosal surface; and (3) are freely dissociable from ENaC. More potentdrugs with longer half-lives on the mucosal surface are needed.

Too little protective surface liquid on other mucosal surfaces is acommon pathophysiology of a number of diseases. For example, inxerostomia (dry mouth) the oral cavity is depleted of liquid due to afailure of the parotid sublingual and submandibular glands to secreteliquid despite continued Na⁺ (ENaC) transport mediated liquid absorptionfrom the oral cavity. Keratoconjunctivitis sira (dry eye) is caused byfailure of lacrimal glands to secrete liquid in the face of continuedNa⁺ dependent liquid absorption on conjunctional surfaces. Inrhinosinusitis, there is an imbalance between mucin secretion andrelative ASL depletion. Failure to secrete Cl— (and liquid) in theproximal small intestine, combined with increased Na⁺ (and liquid)absorption in the terminal Ileum leads to the distal intestinalobstruction syndrome (DIOS). In older patients excessive Na⁺ (andvolume) absorption in the descending colon produces constipation anddiverticulitis.

The published literature includes number of patent applications andgranted patents to Parion Sciences Inc., directed towardpyrazinoylguanidine analogs as sodium channel blockers. Examples of suchpublications include PCT Publication Nos. WO2003/070182, WO2003/070184,WO2004/073629, WO2005/025496, WO2005/016879, WO2005/018644,WO2006/022935, WO2006/023573, WO2006/023617, WO2007/018640,WO2007/146869, WO2008/031028, WO2008/031048, and U.S. Pat. Nos.6,858,614, 6,858,615, 6,903,105, 7,064,129, 7,186,833, 7,189,719,7,192,958, 7,192,959, 7,192,960, 7,241,766, 7,247,636, 7,247,637,7,317,013, 7,332,496, 7,368,447, 7,368,450, 7,368,451, 7,375,102,7,388,013, 7,399,766, 7,410,968, 7,807,834, 7,842,697, and 7,868,010.

There remains a need for novel sodium channel blocking compounds withenhanced potency and effectiveness on mucosal tissues. There alsoremains the need for novel sodium channel blocking compounds thatprovide therapeutic effect, but minimize or eliminate the onset orprogression of hyperkalemia in recipients.

SUMMARY OF THE INVENTION

This invention provides the compound3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide, of the formula:

or a pharmaceutically acceptable salt form thereof. The invention alsoprovides solvates and hydrates, individual stereoisomers, includingoptical isomers (enantiomers and diastereomers) and geometric isomers(cis-/trans-isomerism), mixtures of stereoisomers, and tautomers of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl(2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide, or a pharmaceutically acceptablesalt thereof, as well as pharmaceutical compositions comprising thecompound, or a pharmaceutically acceptable salt thereof, its use inmethods of treatment, and methods for Its preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the advantagesthereof may be readily obtained by reference to the information hereinin conjunction with the following figures:

FIG. 1 is a representative plot of the concentration-effect relationshipof Compound (Ia) on short-circuit current by canine bronchial epithelial(CBE) cells.

FIG. 2 is a plot of the dose-response of Compound (Ia) on sheepmucociliary clearance (MCC) at 4 h post-dose.

FIG. 3 is a plot of the effect of Compound (Ia) and hypertonic saline(HS) on sheep MCC at 4 h post-dose.

FIG. 4 is a plot of the effect Compound (Ia) and HS on sheep MCC at 8 hpost-dose.

FIG. 5 is a plot of the effect of sodium channel blocking of Compound(Ia) on surface liquid retention 0-8 h in the in vitro CBE cell model.

FIG. 6 is a bar graph of the effect of Compound (Ia) on surface liquidretention at 24 hours in the in vitro CBE model.

FIG. 7 is a plot of the effect of ENaC blockers Compound Ia andComparative Example 1 on sheep MCC at 8 hrs.

FIG. 8 is a plot of the effect of ENaC Blockers Compound Ia andComparative Example 1 on sheep plasma potassium levels

FIG. 9 is a plot comparing the activity of Comparative Example 4 andCompound 1a on sheep MCC at 4 h Post-dose.

FIG. 10 is a plot comparing the effect on sheep Plasma K⁺ levels ofComparative Example 4 and Compound Ia.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the following terms are defined as indicated.

“A compound of the invention” means a compound of Formula I or a salt,particularly a pharmaceutically acceptable salt thereof.

“A compound of Formula I” means a compound having the structural formuladesignated herein as Formula I. Compounds of Formula I include solvatesand hydrates (i.e., adducts of a compound of Formula I with a solvent).In those embodiments wherein a compound of Formula I includes one ormore chiral centers, the phrase is intended to encompass each individualstereoisomer including optical isomers (enantiomers and diastereomers)and geometric isomers (cis-/trans-isomerism) and mixtures ofstereoisomers. In addition, compounds of Formula I also includetautomers of the depicted formula(s).

Throughout the description and examples, compounds are named usingstandard IUPAC naming principles, where possible, including the use ofthe ChemDraw Ultra 11.0 software program for naming compounds, sold byCambridgeSoft Corp./PerkinElmer.

In some chemical structure representations where carbon atoms do nothave a sufficient number of attached variables depicted to produce avalence of four, the remaining carbon substituents needed to provide avalence of four should be assumed to be hydrogen. Similarly, in somechemical structures where a bond is drawn without specifying theterminal group, such bond is indicative of a methyl (Me, —CH) group, asis conventional in the art.

In one preferred embodiment, the compound of formula (I) is3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide, having the formula:

or a pharmaceutically acceptable salt thereof.

The compounds of Formula I, may be in the form of a free base or a salt,particularly a pharmaceutically acceptable salt. For a review ofpharmaceutically acceptable salts see Berge et al., J. Pharma Sci.(1977) 66:1-19.

Pharmaceutically acceptable salts formed from inorganic or organic acidsinclude for example, hydrochloride, hydrobromide, hydroiodide, sulfate,bisulfate, nitrate, sulfamate, phosphate, hydrogen phosphate, acetate,trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate,formate, gluconate, succinate, pyruvate, tannate, ascorbate, palmitate,salicylate, stearate, phthalate, alginate, polyglutamate, oxalate,oxaloacetate, saccharate, benzoate, alkyl or aryl sulfonates (e.g.,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateor naphthalenesulfonate) and isothionate; complexes formed with aminoacids such as lysine, arginine, glutamic acid, glycine, serine,threonine, alanine, isoleucine, leucine and the like. The compounds ofthe invention may also be in the form of salts formed from elementalanions such as chlorine, bromine or iodine.

For therapeutic use, salts of active ingredients of the compounds ofFormula I will be pharmaceutically acceptable, i.e. they will be saltsderived from a pharmaceutically acceptable acid. However, salts of acidswhich are not pharmaceutically acceptable may also find use, forexample, in the preparation or purification of a pharmaceuticallyacceptable compound. Trifluoroacetate salts, for example, may find suchuse. All salts, whether or not derived from a pharmaceuticallyacceptable acid, are within the scope of the present invention.

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space. “Diastereomer” refers to a stereoisomer withtwo or more centers of chirality and whose molecules are not mirrorimages of one another. Diastereomers have different physical properties,e.g. melting points, boiling points, spectral properties, andreactivities. Mixtures of diastereomers may separate under highresolution analytical procedures such as electrophoresis andchromatography. “Enantiomers” refer to two stereoisomers of a compoundwhich are non-superimposable mirror Images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., MCGRAW-HILL DICTIONARY OF CHEMICAL TERMS (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,STEREOCHEMISTRY OF ORGANIC COMPOUNDS (1994) John Wiley & Sons, Inc., NewYork.

Many organic compounds exist in optically active forms, i.e., they havethe ability to rotate the plane of plane-polarized light. In describingan optically active compound, the prefixes D and L or R and S are usedto denote the absolute configuration of the molecule about its chiralcenter(s). A specific stereoisomer may also be referred to as anenantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species.

The term “tautomers” refers to a type of stereoisomer in which migrationof a hydrogen atom results in two or more structures. The compounds ofFormula I may exist in different tautomeric forms. One skilled in theart will recognize that amidines, amides, guanidines, ureas, thioureas,heterocycles and the like can exist in tautomeric forms. By way ofexample and not by way of limitation, compounds of Formula I can existin various tautomeric forms as shown below:

All possible tautomeric forms of the amidines, amides, guanidines,ureas, thioureas, heterocycles and the like of all of the embodiments ofFormula I are within the scope of the instant invention. Tautomers existin equilibrium and thus the depiction of a single tautomer in theformulas provided will be understood by those skilled in the art torefer equally to all possible tautomers.

It is to be noted that all enantiomers, diastereomers, and racemicmixtures, tautomers, polymorphs, pseudopolymorphs of compounds withinthe scope of Formula I and pharmaceutically acceptable salts thereof areembraced by the present Invention. All mixtures of such enantiomers anddiastereomers, including enantiomerically enriched mixtures anddiastereomerically enriched mixtures are within the scope of the presentinvention. Enantiomerically enriched mixtures are mixtures ofenantiomers wherein the ratio of the specified enantiomer to thealternative enantiomer is greater than 50:50. More particularly, anenantiomerically enriched mixture comprises at least about 75% of thespecified enantiomer, and preferably at least about 85% of the specifiedenantiomer. In one embodiment, the enantiomerically enriched mixture issubstantially free of the other enantiomer. Similarly,diastereomerically enriched mixtures are mixtures of diastereomerswherein amount of the specified diastereomer is greater than the amountof each alternative diastereomer. More particularly, adiastereomerically enriched mixture comprises at least about 75% of thespecified diastereomer, and preferably at least about 85% of thespecified diastereomer. In one embodiment, the diastereomericallyenriched mixture is substantially free of all other diastereomers. Theterm “substantially free of” will be understood by those skilled in theart to indicate less than a 5% presence of other diastereomers,preferably less than 1%, more preferably less than 0.1%. In otherembodiments no other diastereomers will be present or the amount of anyother diastereomers present will be below the level of detection.Stereoisomers may be separated by techniques known in the art, includinghigh performance liquid chromatography (HPLC) and crystallization ofchiral salts.

A single stereoisomer, e.g. an enantiomer, substantially free of itsstereoisomer may be obtained by resolution of the racemic mixture usinga method such as formation of diastereomers using optically activeresolving agents (“Stereochemistry of Carbon Compounds,” (1962) by E. LEliel, McGraw Hill; Lochmuller, C. H., (1975) J. Chromatogr., 113:(3)283-302). Racemic mixtures of chiral compounds of the invention can beseparated and isolated by any suitable method, including: (1) formationof ionic, diastereomeric salts with chiral compounds and separation byfractional crystallization or other methods, (2) formation ofdiastereomeric compounds with chiral derivatizing reagents, separationof the diastereomers, and conversion to the pure stereoisomers, and (3)separation of the substantially pure or enriched stereoisomers directlyunder chiral conditions.

For illustrative purposes, specific examples of enantiomers of thecompound of formula (I) within the scope of the present inventioninclude, but are not limited to:3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2R,3S,4S,5S)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2R,3S,4S,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4(2-(hexyl((2S,3R,4R,5S)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N(N-4-(4-(2-(hexyl(2R,3S,4R,5S)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazin-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4(2-(hexyl((2S,3R,4S,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)buty)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2R,3R,4S,5S)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3S,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2R,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6chloro-N—(N-(4-(4-(2-(hexyl((2S,3S,4S,5S)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

3,5-diamino-6-chloro-N-(4-(4-(2-(hexyl((2R,3S,4S,5S)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

and 3,5-diamino-6-chloro-N—(N-(4(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

In one embodiment, the present invention provides an enantiomericallyenriched mixture or composition comprising5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide,or a pharmaceutically acceptable salt thereof, as the predominantisomer.

Other embodiments comprise the enantiomerically enriched mixtures orcompositions comprising, respectively, the compounds of formulas (Ia),(Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), and (Il), ora pharmaceutically acceptable salt thereof, as the predominant isomer ineach of their respective mixtures.

In another embodiment, the present invention provides anenantiomerically enriched mixture or composition comprising5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl) carbamimidoyl) pyrazine-2-carboxamide, or apharmaceutically acceptable salt thereof, substantially free of otherisomers.

Four other embodiments comprise the enantiomerically enriched mixturesor compositions comprising, respectively, the compounds of formulas(Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), and(Il), or a pharmaceutically acceptable salt thereof, substantially freeof other isomers in each of their respective mixtures.

A compound of Formula I and pharmaceutically acceptable salts thereofmay exist as different polymorphs or pseudopolymorphs. As used herein,crystalline polymorphism means the ability of a crystalline compound toexist in different crystal structures. The crystalline polymorphism mayresult from differences in crystal packing (packing polymorphism) ordifferences in packing between different conformers of the same molecule(conformational polymorphism). As used herein, crystallinepseudopolymorphism also includes the ability of a hydrate or solvate ofa compound to exist in different crystal structures. Thepseudopolymorphs of the instant invention may exist due to differencesin crystal packing (packing pseudopolymorphism) or due to differences inpacking between different conformers of the same molecule(conformational pseudopolymorphism). The instant invention comprises allpolymorphs and pseudopolymorphs of the compounds of Formula I andpharmaceutically acceptable salts thereof.

A compound of Formula I and pharmaceutically acceptable salts thereofmay also exist as an amorphous solid. As used herein, an amorphous solidis a solid in which there is no long-range order of the positions of theatoms in the solid. This definition applies as well when the crystalsize is two nanometers or less. Additives, including solvents, may beused to create the amorphous forms of the instant invention. The instantinvention, including all pharmaceutical compositions, methods oftreatment, combination products, and uses thereof described herein,comprises all amorphous forms of the compounds of Formula I andpharmaceutically acceptable salts thereof.

Uses

The compounds of the invention exhibit activity as sodium channelblockers. Without being bound by any particular theory, it is believedthat the compounds of the invention may function in vivo by blockingepithelial sodium channels present in mucosal surfaces and therebyreduce the absorption of water by the mucosal surfaces. This effectincreases the volume of protective liquids on mucosal surfaces, andrebalances the system.

As a consequence, the compounds of the invention are useful asmedicaments, particularly for the treatment of clinical conditions forwhich a sodium channel blocker may be indicated. Such conditions includepulmonary conditions such as diseases associated with reversible orirreversible airway obstruction, chronic obstructive pulmonary disease(COPD), including acute exacerbations of COPD, asthma, bronchiectasis(including bronchiectasis due to conditions other than cystic fibrosis),acute bronchitis, chronic bronchitis, post-viral cough, cystic fibrosis,emphysema, pneumonia, panbronchiolitis, and transplant-associatedbronchiolitis, including lung- and bone marrow-transplant associatedbronchiolitis, in a human in need thereof. The compounds of theinvention may also be useful for treating ventilator-associatedtracheobronchitis and/or preventing ventilator-associated pneumonia inventilated patients. The present invention comprises methods fortreating each of these conditions in a mammal in need thereof,preferably in a human in need thereof, each method comprisingadministering to said mammal a pharmaceutically effective amount of acompound of the present invention, or a pharmaceutically acceptable saltthereof. Also provided are (a) a method for reducing exacerbations ofCOPD in a mammal in need thereof; (b) a method for reducingexacerbations of CF in a mammal in need thereof; (c) a method ofimproving lung function (FEV1) in a mammal in need thereof, (d) a methodof improving lung function (FEV1) in a mammal experiencing COPD, (e) amethod of improving lung function (FEV1) in a mammal experiencing CF,(f) a method of reducing airway infections in a mammal in need thereof.

Also provided is a method of stimulating, enhancing or improvingmucociliary clearance in a mammal, the method comprising administeringto a mammal in need thereof a pharmaceutically effective amount of acompound of formula (I), or a pharmaceutically acceptable salt thereof.Mucociliary clearance will be understood to include the naturalmucociliary actions involved in the transfer or clearance of mucus inthe airways, including the self-clearing mechanisms of the bronchi.Therefore, also provided is a method of improving mucus clearance in theairways of a mammal in need thereof.

Additionally, sodium channel blockers may be indicated for the treatmentof conditions which are ameliorated by increased mucosal hydration inmucosal surfaces other than pulmonary mucosal surfaces. Examples of suchconditions include dry mouth (xerostomia), dry skin, vaginal dryness,sinusitis, rhinosinusitis, nasal dehydration, including nasaldehydration brought on by administering dry oxygen, dry eye, Sjogren'sdisease, otitis media, primary ciliary dyskinesia, distal intestinalobstruction syndrome, esophagitis, constipation, and chronicdiverticulitis. The compounds of the invention can also be used forpromoting ocular or corneal hydration.

The compounds of the present invention may also be useful in methods forobtaining a sputum sample from a human. The method may be carried out byadministering a compound of the invention to at least one lung of thepatient, and then inducing and collecting a sputum sample from thathuman.

Accordingly, in one aspect, the present invention provides a method forthe treatment of a condition in a mammal, such as a human, for which asodium channel blocker is indicated.

In other embodiments, the present invention provides each of the methodsdescribed herein with the additional benefit of minimizing oreliminating hyperkalemia in the recipient of the method. Also providedare embodiments comprising each of the methods described herein whereinan Improved therapeutic index is achieved.

The terms “treat”, “treating” and “treatment”, as used herein refers toreversing, alleviating, inhibiting the progress of, or preventing thedisorder or condition or one or more symptoms of such disorder orcondition.

All therapeutic methods described herein are carried out byadministering an effective amount of a compound of the invention, acompound of Formula I or a pharmaceutically acceptable salt thereof, toa subject (typically mammal and preferably human) in need of treatment.

In one embodiment the invention provides a method for the treatment of acondition which is ameliorated by increased mucosal hydration in amammal, particularly a human in need thereof. In one embodiment theinvention provides a method for the treatment of a disease associatedwith reversible or irreversible airway obstruction in a mammal,particularly a human, in need thereof. In one particular embodiment thepresent invention provides a method for the treatment of chronicobstructive pulmonary disease (COPD) in a mammal, particularly a humanin need thereof. In one particular embodiment the present inventionprovides a method for reducing the frequency, severity or duration ofacute exacerbation of COPD or for the treatment of one or more symptomsof acute exacerbation of COPD in a mammal, particularly a human in needthereof. In one embodiment the invention provides a method for thetreatment of asthma in a mammal, particularly a human, in need thereof.In one embodiment the invention provides a method for the treatment ofbronchiectasis (including bronchiectasis due to conditions other thancystic fibrosis) in a mammal, particularly a human, in need thereof. Inone embodiment the invention provides a method for the treatment ofbronchitis, including acute and chronic bronchitis in a mammal,particularly a human, in need thereof. In one embodiment the inventionprovides a method for the treatment of post-viral cough in a mammal,particularly a human, in need thereof. In one embodiment the inventionprovides a method for the treatment of cystic fibrosis in a mammal,particularly a human, in need thereof. In one embodiment the inventionprovides a method for the treatment of emphysema in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of pneumonia in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of panbronchiolitis in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of transplant-associatedbronchiolitis, including lung- and bone marrow-transplant associatedbronchiolitis in a mammal, particularly a human in need thereof. In oneembodiment the invention provides a method for treatingventilator-associated tracheobronchitis and/or preventingventilator-associated pneumonia in a ventilated human in need thereof.

This invention provides specific methods for treating a disease selectedfrom the group of reversible or irreversible airway obstruction, chronicobstructive pulmonary disease (COPD), asthma, bronchiectasis (includingbronchiectasis due to conditions other than cystic fibrosis), acutebronchitis, chronic bronchitis, post-viral cough, cystic fibrosis,emphysema, pneumonia, panbronchiolitis, transplant-associatebronchiolitis, and ventilator-associated tracheobronchitis or preventingventilator-associated pneumonia in a human in need thereof, each methodcomprising administering to said human an effective amount of a compoundof formula 1(a), or a pharmaceutically acceptable salt thereof. Infurther embodiments for each method of treatment, the pharmaceuticallyacceptable salt form is a hydrochloride salt or a hydroxynaphthoate saltof the compound of formula (1a). In another embodiment within eachmethod of treatment, the freebase of the compound of formula (1a) isused.

In one embodiment the invention provides a method for the treatment ofdry mouth (xerostomia) in a mammal, particularly a human in needthereof. In one embodiment the invention provides a method for thetreatment of dry skin in a mammal, particularly a human in need thereof.In one embodiment the invention provides a method for the treatment ofvaginal dryness in a mammal, particularly a human in need thereof. Inone embodiment the invention provides a method for the treatment ofsinusitis, rhinosinusitis, or nasal dehydration, including nasaldehydration brought on by administering dry oxygen, in a mammal,particularly a human in need thereof. In one embodiment the inventionprovides a method for the treatment of dry eye, or Sjogren's disease, orpromoting ocular or corneal hydration in a mammal, particularly a humanin need thereof. In one embodiment the invention provides a method forthe treatment of otitis media in a mammal, particularly a human in needthereof. In one embodiment the invention provides a method for thetreatment of primary ciliary dyskinesia, in a mammal, particularly ahuman in need thereof. In one embodiment the invention provides a methodfor the treatment of distal intestinal obstruction syndrome,esophagitis, constipation, or chronic diverticulitis in a mammal,particularly a human in need thereof.

There is also provided a compound of the invention for use in medicaltherapy, particularly for use in the treatment of condition in a mammal,such as a human, for which a sodium channel blocker is indicated. Alltherapeutic uses described herein are carried out by administering aneffective amount of a compound of the invention to the subject in needof treatment. In one embodiment there is provided a compound of theinvention for use in the treatment of a pulmonary condition such as adisease associated with reversible or irreversible airway obstruction ina mammal, particularly a human, in need thereof. In one particularembodiment there is provided a compound of the invention for use in thetreatment of chronic obstructive pulmonary disease (COPD) in a mammal,particularly a human in need thereof. In one embodiment, there isprovided a compound of the invention for use in reducing the frequency,severity or duration of acute exacerbation of COPD or for the treatmentof one or more symptoms of acute exacerbation of COPD, in a mammal,particularly a human, in need thereof. In one embodiment there isprovided a compound of the invention for use in the treatment of asthmain a mammal, particularly a human, in need thereof. In one embodimentthere is provided a compound for use in the treatment of bronchiectasis,including bronchiectasis due to conditions other than cystic fibrosis,or bronchitis, including acute bronchitis and chronic bronchitis, in amammal, particularly a human, in need thereof. In one embodiment thereis provided a compound for use in the treatment of post-viral cough, ina mammal, particularly a human, in need thereof. In one embodiment thereis provided a compound for use in the treatment of cystic fibrosis in amammal, particularly a human in need thereof. In one embodiment there isprovided a compound of the invention for use in the treatment ofemphysema in a mammal, particularly a human, in need thereof. In oneembodiment there is provided a compound of the invention for use in thetreatment of pneumonia in a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound of the inventionfor use in the treatment of panbronchiolitis or transplant-associatedbronchiolitis, including lung- and bone marrow-transplant associatedbronchiolitis in a mammal, particularly a human, in need thereof. In oneembodiment there is provided a compound of the invention for use in thetreatment of ventilator-associated tracheobronchitis or preventingventilator-associated pneumonia in a ventilated human in need thereof.

In one embodiment there is provided a compound of the invention for usein the treatment of a condition ameliorated by increased mucosalhydration in mucosal surfaces of a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound for use in thetreatment of dry mouth (xerostomia) in a mammal, particularly a human,in need thereof. In one embodiment there is provided a compound for usein the treatment of dry skin in a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound for use in thetreatment of vaginal dryness in a mammal, particularly a human in needthereof. In one embodiment there is provided a compound of the inventionfor use in the treatment of sinusitis, rhinosinusitis, or nasaldehydration, including nasal dehydration brought on by administering dryoxygen in a mammal, particularly a human, in need thereof. In oneembodiment there is provided a compound of the invention for use in thetreatment of dry eye, or Sjogren's disease or promoting ocular orcorneal hydration in a mammal, particularly a human, in need thereof. Inone embodiment there is provided a compound of the invention for use inthe treatment of otitis media in a mammal, particularly a human, in needthereof. In one embodiment there is provided a compound of the inventionfor use in the treatment of primary ciliary dyskinesia in a mammal,particularly a human, in need thereof. In one embodiment there isprovided a compound of the invention for use in the treatment of distalintestinal obstruction syndrome, esophagitis, constipation, or chronicdiverticulitis in a mammal, particularly a human, in need thereof.

The present invention also provides the use of a compound of theinvention in the manufacture of a medicament for the treatment of acondition in a mammal, such as a human, for which a sodium channelblocker is indicated. In one embodiment is provided the use of acompound of the invention in the manufacture of a medicament for thetreatment of diseases associated with reversible or irreversible airwayobstruction, chronic obstructive pulmonary disease (COPD), acuteexacerbations of COPD, asthma, bronchiectasis (including bronchiectasisdue to conditions other than cystic fibrosis), bronchitis (includingacute bronchitis and chronic bronchitis), post-viral cough, cysticfibrosis, emphysema, pneumonia, panbronchiolitis, transplant-associatedbronchiolitis, (including lung- and bone marrow-transplant associatedbronchiolitis), ventilator-associated tracheobronchitis or preventingventilator-associated pneumonia.

In one particular embodiment is provided the use of a compound of theinvention in the manufacture of a medicament for the treatment of acondition ameliorated by increased mucosal hydration in mucosalsurfaces, treatment of dry mouth (xerostomia), dry skin, vaginaldryness, sinusitis, rhinosinusitis, nasal dehydration, including nasaldehydration brought on by administering dry oxygen, treatment of dryeye, Sjogren's disease, promoting ocular or corneal hydration, treatmentof otitis media, primary ciliary dyskinesia, distal intestinalobstruction syndrome, esophagitis, constipation, or chronicdiverticulitis

The terms “effective amount”, “pharmaceutically effective amount”,“effective dose”, and “pharmaceutically effective dose” as used herein,refer to an amount of compound of the invention which is sufficient inthe subject to which it is administered, to elicit the biological ormedical response of a cell culture, tissue, system, or mammal (includinghuman) that is being sought, for instance by a researcher or clinician.The term also includes within its scope, amounts effective to enhancenormal physiological function. In one embodiment, the effective amountis the amount needed to provide a desired level of drug in thesecretions and tissues of the airways and lungs, or alternatively, inthe bloodstream of a subject to be treated to give an anticipatedphysiological response or desired biological effect when such acomposition is administered by inhalation. For example an effectiveamount of a compound of the invention for the treatment of a conditionfor which a sodium channel blocker is indicated is sufficient in thesubject to which it is administered to treat the particular condition.In one embodiment an effective amount is an amount of a compound of theinvention which is sufficient for the treatment of COPD or cysticfibrosis in a human.

The precise effective amount of the compounds of the invention willdepend on a number of factors including but not limited to the species,age and weight of the subject being treated, the precise conditionrequiring treatment and its severity, the bioavailability, potency, andother properties of the specific compound being administered, the natureof the formulation, the route of administration, and the deliverydevice, and will ultimately be at the discretion of the attendantphysician or veterinarian. Further guidance with respect to appropriatedose may be found in considering conventional dosing of other sodiumchannel blockers, such as amiloride, with due consideration also beinggiven to any differences in potency between amiloride and the compoundsof the present invention.

A pharmaceutically effective dose administered topically to the airwaysurfaces of a subject (e.g., by inhalation) of a compound of theinvention for treatment of a 70 kg human may be in the range of fromabout 10 ng to about 10 mg. In another embodiment, the pharmaceuticallyeffective dose may be from about 0.1 to about 1000 μg. Typically, thedaily dose administered topically to the airway surfaces will be anamount sufficient to achieve dissolved concentration of active agent onthe airway surfaces of from about 10⁻⁹, 10⁻⁸, or 10⁻⁷ to about 10⁻⁴,10⁻³, 10⁻², or 10⁻¹ Moles/liter, more preferably from about 10⁻⁹ toabout 10⁻⁴ Moles/liter. The selection of the specific dose for a patientwill be determined by the attendant physician, clinician or veterinarianof ordinary skill in the art based upon a number of factors includingthose noted above. In one particular embodiment the dose of a compoundof the invention for the treatment of a 70 kg human will be in the rangeof from about 10 nanograms (ng) to about 10 mg. In another embodiment,the effective dose would be from about 0.1 μg to about 1,000 μg. In oneembodiment, the dose of a compound of the invention for the treatment ofa 70 kg human will be in the range of from about 0.5 μg to about 0.5 mg.In a further embodiment the dose will be from about 0.5 μg to about 60μg. In another embodiment, the pharmaceutically effective dose will befrom about 1 to about 10 μg. In another embodiment, the pharmaceuticallyeffective dose will be from about 5 μg to about 50 μg. Anotherembodiment will have an effective dose of from about 10 μg to about 40μg. In two further embodiments, the pharmaceutically effective dose willbe from about 15 μg to about 50 μg from about 15 μg to about 30 μg,respectively. It will be understood that in each of these dose ranges,all Incremental doses in the range are included. For instance, the0.5-50 μg range includes individual doses of: 0.5 μg, 0.6 μg, 0.7 μg,0.8 μg, 0.9 μg, 1.0 μg, 1.1 μg, 1.2 μg, 1.3 μg, 1.4 μg, 1.5 μg, 1.6 μg,1.7 μg, 1.8 μg, 1.9 μg, 2.0 μg, 2.1 μg, 2.2 μg, 2.3 μg, 2.4 μg, 2.5 μg,2.6 μg, 2.7 μg, 2.8 μg, 2.9 μg, 3.0 μg, 3.1 μg, 3.2 μg, 3.3 μg, 3.4 μg,3.5 μg, 3.6 μg, 3.7 μg, 3.8 μg, 3.9 μg, 4.0 μg, 4.1 μg, 4.2 μg, 4.3 μg,4.4 μg, 4.5 μg, 4.6 μg, 4.7 μg, 4.8 μg, 4.9 μg, 5.0 μg, 5.1 μg, 5.2 μg,5.3 μg, 5.4 μg, 5.5 μg, 5.6 μg, 5.7 μg, 5.8 μg, 5.9 μg, 6.0 μg, 6.1 μg,6.2 μg, 6.3 μg, 6.4 μg, 6.5 μg, 6.6 μg, 6.7 μg, 6.8 μg, 6.9 μg, 7.0 μg,7.1 μg, 7.2 μg, 7.3 μg, 7.4 μg, 7.5 μg, 7.6 μg, 7.7 μg, 7.8 μg, 7.9 μg,8.0 μg, 8.1 μg, 8.2 μg, 8.3 μg, 8.4 μg, 8.5 μg, 8.6 μg, 8.7 μg, 8.8 μg,8.9 μg, 9.0 μg, 9.1 μg, 9.2 μg, 9.3 μg, 9.4 μg, 9.5 μg, 9.6 μg, 9.7 μg,9.8 μg, 9.9 μg, 10.0 μg, 10.1 μg, 10.2 μg, 10.3 μg, 10.4 μg, 10.5 μg,10.6 μg, 10.7 μg, 10.8 μg, 10.9 μg, 11.0 μg, 11.1 μg, 11.2 μg, 11.3 μg,11.4 μg, 11.5 μg, 11.6 μg, 11.7 μg, 11.8 μg, 11.9 μg, 12.0 μg, 12.1 μg,12.2 μg, 12.3 μg, 12.4 μg, 12.5 μg, 12.6 μg, 12.7 μg, 12.8 μg, 12.9 μg,13.0 μg, 13.1 μg, 13.2 μg, 13.3 μg, 13.4 μg, 13.5 μg, 13.6 μg, 13.7 μg,13.8 μg, 13.9 μg, 14.0 μg, 14.1 μg, 14.2 μg, 14.3 μg, 14.4 μg, 14.5 μg,14.6 μg, 14.7 μg, 14.8 μg, 14.9 μg, 15.0 μg, 15.1 μg, 15.2 μg, 15.3 μg,15.4 μg, 15.5 μg, 15.6 μg, 15.7 μg, 15.8 μg, 15.9 μg, 16.0 μg, 16.1 μg,16.2 μg, 16.3 μg, 16.4 μg, 16.5 μg, 16.6 μg, 16.7 μg, 16.8 μg, 16.9 μg,17.0 μg, 17.1 μg, 17.2 μg, 17.3 μg, 17.4 μg, 17.5 μg, 17.6 μg, 17.7 μg,17.8 μg, 17.9 μg, 18.0 μg, 18.1 μg, 18.2 μg, 18.3 μg, 18.4 μg, 18.5 μg,18.6 μg, 18.7 μg, 18.8 μg, 18.9 μg, 19.0 μg, 19.1 μg, 19.2 μg, 19.3 μg,19.4 μg, 19.5 μg, 19.6 μg, 19.7 μg, 19.8 μg, 19.9 μg, 20.0 μg, 20.1 μg,20.2 μg, 20.3 μg, 20.4 μg, 20.5 μg, 20.6 μg, 20.7 μg, 20.8 μg, 20.9 μg,21.0 μg, 21.1 μg, 21.2 μg, 21.3 μg, 21.4 μg, 21.5 μg, 21.6 μg, 21.7 μg,21.8 μg, 21.9 μg, 22.0 μg, 22.1 μg, 22.2 μg, 22.3 μg, 22.4 μg, 22.5 μg,22.6 μg, 22.7 μg, 22.8 μg, 22.9 μg, 23.0 μg, 23.1 μg, 23.2 μg, 23.3 μg,23.4 μg, 23.5 μg, 23.6 μg, 23.7 μg, 23.8 μg, 23.9 μg, 24.0 μg, 24.1 μg,24.2 μg, 24.3 μg, 24.4 μg, 24.5 μg, 24.6 μg, 24.7 μg, 24.8 μg, 24.9 μg,25.0 μg, 25.1 μg, 25.2 μg, 25.3 μg, 25.4 μg, 25.5 μg, 25.6 μg, 25.7 μg,25.8 μg, 25.9 μg, 26.0 μg, 26.1 μg, 26.2 μg, 26.3 μg, 26.4 μg, 26.5 μg,26.6 μg, 26.7 μg, 26.8 μg, 26.9 μg, 27.0 μg, 27.1 μg, 27.2 μg, 27.3 μg,27.4 μg, 27.5 μg, 27.6 μg, 27.7 μg, 27.8 μg, 27.9 μg, 28.0 μg, 28.1 μg,28.2 μg, 28.3 μg, 28.4 μg, 28.5 μg, 28.6 μg, 28.7 μg, 28.8 μg, 28.9 μg,29.0 μg, 29.1 μg, 29.2 μg, 29.3 μg, 29.4 μg, 29.5 μg, 29.6 μg, 29.7 μg,29.8 μg, 29.9 μg, 30.0 μg, 30.1 μg, 30.2 μg, 30.3 μg, 30.4 μg, 30.5 μg,30.6 μg, 30.7 μg, 30.8 μg, 30.9 μg, 31.0 μg, 31.1 μg, 31.2 μg, 31.3 μg,31.4 μg, 31.5 μg, 31.6 μg, 31.7 μg, 31.8 μg, 31.9 μg, 32.0 μg, 32.1 μg,32.2 μg, 32.3 μg, 32.4 μg, 32.5 μg, 32.6 μg, 32.7 μg, 32.8 μg, 32.9 μg,33.0 μg, 33.1 μg, 33.2 μg, 33.3 μg, 33.4 μg, 33.5 μg, 33.6 μg, 33.7 μg,33.8 μg, 33.9 μg, 34.0 μg, 34.1 μg, 34.2 μg, 34.3 μg, 34.4 μg, 34.5 μg,34.6 μg, 34.7 μg, 34.8 μg, 34.9 μg, 35.0 μg, 35.1 μg, 35.2 μg, 35.3 μg,35.4 μg, 35.5 μg, 35.6 μg, 35.7 μg, 35.8 μg, 35.9 μg, 36.0 μg, 36.1 μg,36.2 μg, 36.3 μg, 36.4 μg, 36.5 μg, 36.6 μg, 36.7 μg, 38.8 μg, 36.9 μg,37.0 μg, 37.1 μg, 37.2 μg, 37.3 μg, 37.4 μg, 37.5 μg, 37.6 μg, 37.7 μg,37.8 μg, 37.9 μg, 38.0 μg, 38.1 μg, 38.2 μg, 38.3 μg, 38.4 μg, 38.5 μg,38.6 μg, 38.7 μg, 38.8 μg, 38.9 μg, 39.0 μg, 39.1 μg, 39.2 μg, 39.3 μg,39.4 μg, 39.5 μg, 39.6 μg, 39.7 μg, 39.8 μg, 39.9 μg, 40.0 μg, 40.1 μg,40.2 μg, 40.3 μg, 40.4 μg, 40.5 μg, 40.6 μg, 40.7 μg, 40.8 μg, 40.9 μg,41.0 μg, 41.1 μg, 41.2 μg, 41.3 μg, 41.4 μg, 41.5 μg, 41.6 μg, 41.7 μg,41.8 μg, 41.9 μg, 42.0 μg, 42.1 μg, 42.2 μg, 42.3 μg, 42.4 μg, 42.5 μg,42.6 μg, 42.7 μg, 42.8 μg, 42.9 μg, 43.0 μg, 43.1 μg, 43.2 μg, 43.3 μg,43.4 μg, 43.5 μg, 43.6 μg, 43.7 μg, 43.8 μg, 43.9 μg, 44.0 μg, 44.1 μg,44.2 μg, 44.3 μg, 44.4 μg, 44.5 μg, 44.6 μg, 44.7 μg, 44.8 μg, 44.9 μg,45.0 μg, 45.1 μg, 45.2 μg, 45.3 μg, 45.4 μg, 45.5 μg, 45.6 μg, 45.7 μg,45.8 μg, 45.9 μg, 46.0 μg, 46.1 μg, 46.2 μg, 46.3 μg, 46.4 μg, 46.5 μg,46.6 μg, 46.7 μg, 46.8 μg, 46.9 μg, 47.0 μg, 47.1 μg, 47.2 μg, 47.3 μg,47.4 μg, 47.5 μg, 47.6 μg, 47.7 μg, 47.8 μg, 47.9 μg, 48.0 μg, 48.1 μg,48.2 μg, 48.3 μg, 48.4 μg, 48.5 μg, 48.6 μg, 48.7 μg, 48.8 μg, 38.9 μg,49.0 μg, 49.1 μg, 49.2 μg, 49.3 μg, 49.4 μg, 49.5 μg, 49.6 μg, 49.7 μg,49.8 μg, 39.9 μg, and 50 μg.

The foregoing suggested doses may be adjusted using conventional dosecalculations if the compound is administered via a different route.Determination of an appropriate dose for administration by other routesis within the skill of those in the art in light of the foregoingdescription and the general knowledge in the art.

Delivery of an effective amount of a compound of the invention mayentail delivery of a single dosage form or multiple unit doses which maybe delivered contemporaneously or separate in time over a designatedperiod, such as 24 hours. A dose of a compound of the invention (aloneor in the form of a composition comprising the same) may be administeredfrom one to ten times per day. Typically, a compound of the invention(alone or in the form of a composition comprising the same) will beadministered four, three, two, or once per day (24 hours).

The compounds of formula (I) of the present invention are also usefulfor treating airborne infections. Examples of airborne infectionsinclude, for example, RSV. The compounds of formula (I) of the presentinvention are also useful for treating an anthrax infection. The presentinvention relates to the use of the compounds of formula (I) of thepresent invention for prophylactic, post-exposure prophylactic,preventive or therapeutic treatment against diseases or conditionscaused by pathogens. In a preferred embodiment, the present inventionrelates to the use of the compounds of formula (I) for prophylactic,post-exposure prophylactic, preventive or therapeutic treatment againstdiseases or conditions caused by pathogens which may be used inbioterrorism.

In recent years, a variety of research programs and biodefense measureshave been put into place to deal with concerns about the use ofbiological agents in acts of terrorism. These measures are intended toaddress concerns regarding bioterrorism or the use of microorganisms orbiological toxins to kill people, spread fear, and disrupt society. Forexample, the National Institute of Allergy and Infectious Diseases(NIAID) has developed a Strategic Plan for Biodefense Research whichoutlines plans for addressing research needs in the broad area ofbioterrorism and emerging and reemerging infectious diseases. Accordingto the plan, the deliberate exposure of the civilian population of theUnited States to Bacillus anthracis spores revealed a gap in thenation's overall preparedness against bioterrorism. Moreover, the reportdetails that these attacks uncovered an unmet need for tests to rapidlydiagnose, vaccines and immunotherapies to prevent, and drugs andbiologics to cure disease caused by agents of bioterrorism.

Much of the focus of the various research efforts has been directed tostudying the biology of the pathogens identified as potentiallydangerous as bioterrorism agents, studying the host response againstsuch agents, developing vaccines against Infectious diseases, evaluatingthe therapeutics currently available and under investigation againstsuch agents, and developing diagnostics to identify signs and symptomsof threatening agents. Such efforts are laudable but, given the largenumber of pathogens which have been identified as potentially availablefor bioterrorism, these efforts have not yet been able to providesatisfactory responses for all possible bioterrorism threats.Additionally, many of the pathogens identified as potentially dangerousas agents of bioterrorism do not provide adequate economic incentivesfor the development of therapeutic or preventive measures by industry.Moreover, even if preventive measures such as vaccines were availablefor each pathogen which may be used in bioterrorism, the cost ofadministering all such vaccines to the general population isprohibitive.

Until convenient and effective treatments are available against everybioterrorism threat, there exists a strong need for preventative,prophylactic or therapeutic treatments which can prevent or reduce therisk of infection from pathogenic agents.

The present invention provides such methods of prophylactic treatment.In one aspect, a prophylactic treatment method is provided comprisingadministering a prophylactically effective amount of the compounds offormula (I) to an individual in need of prophylactic treatment againstinfection from one or more airborne pathogens. A particular example ofan airborne pathogen is anthrax.

In another aspect, a prophylactic treatment method is provided forreducing the risk of infection from an airborne pathogen which can causea disease in a human, said method comprising administering an effectiveamount of the compounds of formula (I) to the lungs of the human who maybe at risk of infection from the airborne pathogen but is asymptomaticfor the disease, wherein the effective amount of a sodium channelblocker and osmolye are sufficient to reduce the risk of infection inthe human. A particular example of an airborne pathogen is anthrax.

In another aspect, a post-exposure prophylactic treatment or therapeutictreatment method is provided for treating infection from an airbornepathogen comprising administering an effective amount of the compoundsof formula (I) to the lungs of an Individual in need of such treatmentagainst infection from an airborne pathogen. The pathogens which may beprotected against by the prophylactic post exposure, rescue andtherapeutic treatment methods of the invention include any pathogenswhich may enter the body through the mouth, nose or nasal airways, thusproceeding into the lungs. Typically, the pathogens will be airbornepathogens, either naturally occurring or by aerosolization. Thepathogens may be naturally occurring or may have been introduced intothe environment intentionally after aerosolization or other method ofintroducing the pathogens into the environment. Many pathogens which arenot naturally transmitted in the air have been or may be aerosolized foruse in bioterrorism. The pathogens for which the treatment of theinvention may be useful includes, but is not limited to, category A, Band C priority pathogens as set forth by the NIAID. These categoriescorrespond generally to the lists compiled by the Centers for DiseaseControl and Prevention (CDC). As set up by the CDC, Category A agentsare those that can be easily disseminated or transmittedperson-to-person, cause high mortality, with potential for major publichealth impact. Category B agents are next in priority and include thosethat are moderately easy to disseminate and cause moderate morbidity andlow mortality. Category C consists of emerging pathogens that could beengineered for mass dissemination in the future because of theiravailability, ease of production and dissemination and potential forhigh morbidity and mortality. Particular examples of these pathogens areanthrax and plague. Additional pathogens which may be protected againstor the Infection risk therefrom reduced include influenza viruses,rhinoviruses, adenoviruses and respiratory syncytial viruses, and thelike. A further pathogen which may be protected against is thecoronavirus which is believed to cause severe acute respiratory syndrome(SARS).

The present invention also relates to the use of sodium channel blockersof Formula I, or a pharmaceutically acceptable salt thereof, forpreventing, mitigating, and/or treating deterministic health effects tothe respiratory tract caused by exposure to radiological materials,particularly respirable aerosols containing radionuclides from nuclearattacks, such as detonation of radiological dispersal devices (RDD), oraccidents, such as nuclear power plant disasters. As such, providedherein is a method for preventing, mitigating, and/or treatingdeterministic health effects to the respiratory tract and/or otherbodily organs caused by respirable aerosols containing radionuclides ina recipient in need thereof, including in a human in need thereof, saidmethod comprising administering to said human an effective amount of acompound of Formula (I), or a pharmaceutically acceptable salt thereof.

A major concern associated with consequence management planning forexposures of members of the public to respirable aerosols containingradionuclides from nuclear attacks, such as detonation of radiologicaldispersal devices (RDD), or accidents, such as nuclear power plantdisasters is how to prevent, mitigate or treat potential deterministichealth effects to the respiratory tract, primarily the lung. It isnecessary to have drugs, techniques and procedures, and trainedpersonnel prepared to manage and treat such highly internallycontaminated individuals.

Research has been conducted to determine ways in which to prevent,mitigate or treat potential damage to the respiratory tract and variousorgans in the body that is caused by internally deposited radionuclides.To date, most of the research attention has focused on strategiesdesigned to mitigate health effects from internally depositedradionuclides by accelerating their excretion or removal. Thesestrategies have focused on soluble chemical forms that are capable ofreaching the blood stream and are deposited at remote systemic sitesspecific to a given radioelement. Such approaches will not work in caseswhere the deposited radionuclide is in relatively insoluble form.Studies have shown that many, if not most of the physicochemical formsof dispersed radionuclides from RDDs, will be in relatively insolubleform.

The only method known to effectively reduce the radiation dose to thelungs from inhaled insoluble radioactive aerosols is bronchoalveolarlavage or BAL. This technique, which was adapted from that already inuse for the treatment of patients with alveolar proteinosis, has beenshown to be a safe, repeatable procedure, even when performed over anextended period of time. Although there are variations in procedure, thebasic method for BAL is to anaesthetize the subject, followed by theslow introduction of isotonic saline into a single lobe of the lunguntil the function residual capacity is reached. Additional volumes arethen added and drained by gravity.

The results of studies using BAL on animals indicate that about 40% ofthe deep lung content can be removed by a reasonable sequence of BALs.In some studies, there was considerable variability among animals in theamount of radionuclide recovered. The reasons for the variability arecurrently not understood.

Further, based on a study on animals, it is believed that a significantdose reduction from BAL therapy results in mitigation of health effectsdue to inhalation of insoluble radionuclides. In the study, adult dogsinhaled insoluble ¹⁴⁴Ce-FAP particles. Two groups of dogs were givenlung contents of ¹⁴⁴Ce known to cause radiation pneumonitis andpulmonary fibrosis (about 2 MBq/kg body mass), with one group beingtreated with 10 unilateral lavages between 2 and 56 days after exposure,the other untreated. A third group was exposed at a level of ⁴⁴Cecomparable to that seen in the BAL-treated group after treatment (about1 MBq/kg), but these animals were untreated. All animals were allowed tolive their lifespans, which extended to 16 years. Because there isvariability in initial lung content of ¹⁴⁴Ce among the dogs in eachgroup, the dose rates and cumulative doses for each group overlap.Nevertheless, the effect of BAL In reducing the risk frompneumonitis/fibrosis was evident from the survival curves. In theuntreated dogs with lung contents of 1.5-2.5 MBq/kg, the mean survivaltime was 370±65 d. For the treated dogs, the mean survival was 1270±240d, which was statistically significantly different. The third group,which received lung contents of ¹⁴⁴Ce of 0.6-1.4 MBq had a mean survivaltime of 1800±230, which was not statistically different from the treatedgroup. Equally important to the increased survival, the dogs in thehigh-dose untreated group died from deterministic effects to lung(pneumonitis/fibrosis) while the treated dogs did not. Instead, thetreated dogs, like the dogs in the low-dose untreated group, mostly hadlung tumors (hemangiosarcoma or carcinoma). Therefore, the reduction indose resulting from BAL treatment appears to have produced biologicaleffects in lung that were predictable based on the radiation doses thatthe lungs received.

Based on these results, it is believed that decreasing the residualradiological dose further by any method or combination of methods forenhancing the clearance of particles from the lung would furtherdecrease the probability of health effects to lung. However, BAL is aprocedure that has many drawbacks. BAL is a highly invasive procedurethat must be performed at specialized medical centers by trainedpulmonologists. As such, a BAL procedure is expensive. Given thedrawbacks of BAL, it is not a treatment option that would be readily andimmediately available to persons in need of accelerated removal ofradioactive particles, for example, in the event of a nuclear attack. Inthe event of a nuclear attack or a nuclear accident, immediate andrelatively easily administered treatment for persons who have beenexposed or who are at risk of being exposed is needed. Sodium channelblockers administered as an inhalation aerosol have been shown torestore hydration of airway surfaces. Such hydration of airway surfacesaids in clearing accumulated mucus secretions and associated particulatematter from the lung. As such, without being bound by any particulartheory, it is believed that sodium channel blockers can be used toaccelerate the removal of radioactive particles from airway passages.

As discussed above, the greatest risk to the lungs following aradiological attack, such as a dirty bomb, results from the inhalationand retention of insoluble radioactive particles. As a result ofradioactive particle retention, the cumulative exposure to the lung issignificantly increased, ultimately resulting in pulmonaryfibrosis/pneumonitis and potentially death. Insoluble particles cannotbe systemically cleared by chelating agents because these particles arenot in solution. To date, the physical removal of particulate matterthrough BAL is the only therapeutic regimen shown to be effective atmitigating radiation-induced lung disease. As discussed above, BAL isnot a realistic treatment solution for reducing the effects ofradioactive particles that have been inhaled into the body. As such, itis desirable to provide a therapeutic regimen that effectively aids inclearing radioactive particles from airway passages and that, unlikeBAL, is relatively simple to administer and scalable in a large-scaleradiation exposure scenario. In addition, it is also desirable that thetherapeutic regimen be readily available to a number of people in arelatively short period of time.

In an aspect of the present invention, a method for preventing,mitigating, and/or treating deterministic health effects to therespiratory tract and/or other bodily organs caused by respirableaerosols containing radionuclides comprises administering an effectiveamount of a sodium channel blocker of Formula I or a pharmaceuticallyacceptable salt thereof to an individual in need. In a feature of thisaspect, the sodium channel blocker is administered in conjunction withan osmolyte. With further regard to this feature, the osmolyte ishypertonic saline (HS). In a further feature, the sodium channel blockerand the osmolyte are administered in conjunction with an ion transportmodulator. With further regard to this feature, the ion transportmodulator may be selected from the group consisting of 1-agonists, CFTRpotentiators, purinergic receptor agonists, lubiprostones, and proteaseinhibitors. In another feature of this aspect, the radionuclides areselected from the group consisting of Colbalt-60, Cesium-137,Iridium-192, Radium-226, Phospohrus-32, Strontium-89 and 90, Iodine-125,Thallium-201, Lead-210, Thorium-234, Uranium-238, Plutonium, Cobalt-58,Chromium-51, Americium, and Curium. In a further feature, theradionuclides are from a radioactive disposal device. In yet anotherfeature, the sodium channel blocker or pharmaceutically acceptable saltthereof is administered in an aerosol suspension of respirable particleswhich the Individual inhales. In an additional feature, the sodiumchannel blocker or a pharmaceutically acceptable salt thereof isadministered post-exposure to the radionuclides.

Compositions

While it is possible for a compound of the invention to be administeredalone, in some embodiments it is preferable to present it in the form ofa composition, particularly a pharmaceutical composition (formulation).Thus, in another aspect, the invention provides compositions, andparticularly pharmaceutical compositions (such as an inhalablepharmaceutical composition) comprising a pharmaceutically effectiveamount of a compound of the invention as an active ingredient, and apharmaceutically acceptable excipient, diluent or carrier. The term“active ingredient” as employed herein refers to any compound of theinvention or combination of two or more compounds of the invention in apharmaceutical composition. Also provided are specific embodiments inwhich a pharmaceutical composition comprises a pharmaceuticallyeffective amount of a compound of Formulas (I), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), and (Il), or apharmaceutically acceptable salt thereof., independently or incombination, and a pharmaceutically acceptable excipient, diluent orcarrier.

In some embodiments, the pharmaceutical composition comprises apharmaceutically effective amount of a compound of Formulas (I), (Ia),(Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ii), (Ik), and (Il), ora pharmaceutically acceptable salt thereof., independently or incombination, in a diluent. In separate embodiments, the pharmaceuticalcomposition comprises a pharmaceutically effective amount of a compoundof Formulas (I), (Ia), (Ib), (Ic), (id), (Ie), (If), (Ig), (Ih), (Ii),(Ij), (Ik), and (Il), or a pharmaceutically acceptable salt thereof, inhypertonic saline, sterile water, and hypertonic saline, respectively,wherein the saline concentration can be as described herein. In oneembodiment the saline concentration is 0.17% w/v and in another it is2.8% w/v.

Also provided is a kit comprising i) a pharmaceutically effective amountof a compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),(Ih), (Ii), (Ij), (Ik), and (Il), or a pharmaceutically acceptable saltthereof; ii) one or more pharmaceutically acceptable excipients,carriers, or diluents; iii) instructions for administering the compoundof group i) and the excipients, carriers, or diluents of group ii) to asubject in need thereof; and; iv) a container. A subject in need thereofincludes any subject in need of the methods of treatment describedherein, particularly including a human subject in need thereof. Furtherembodiments also comprise an aerosolization device selected from thegroup of a nebulizer, including vibrating mesh nebulizers and jetnebulizers, a dry powder inhaler, including active and passive drypowder inhalers, and a metered dose inhaler, including pressurized, drypowder, and soft mist metered dose inhalers.

In one embodiment a kit comprises i) from about 10 ng to about 10 mg ofa compound of Formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig),(Ih), (Ii), (Ij), (Ik), and (Il), or a pharmaceutically acceptable saltthereof, per dose; ii) from about 1 to about 5 mL of diluent per dose;iii) instructions for administering the compound of group i) and thediluent of group ii) to a subject in need thereof; and; iv) a container.In a further embodiment, the diluent is from about 1 to about 5 mL of asaline solution, as described herein, per dose. In a further embodiment,the diluent is from about 1 to about 5 mL of a hypotonic saline solutionper dose. In another embodiment, the diluent is from about 1 to about 5mL of a hypertonic saline solution per dose. In a still furtherembodiment, the diluent is from about 1 to about 5 mL of sterile waterper dose.

Also provided is a kit comprising i) a solution comprising apharmaceutically effective amount of a compound of Formula (I), (Ia),(Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), and (Il), ora pharmaceutically acceptable salt thereof; dissolved in apharmaceutically acceptable diluent; iii) instructions for administeringthe solution of group i) to a subject in need thereof; and iii) acontainer.

Also provided is a kit comprising i) a solution comprising from about 10ng to about 10 mg of a compound of Formula (I), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), and (Il), or apharmaceutically acceptable salt thereof; dissolved in apharmaceutically acceptable diluent; iii) instructions for administeringthe solution of group i) to a subject in need thereof; and iii) acontainer. In a further embodiment, the diluent is from about 1 to about5 mL of a saline solution, as described herein, per dose.

Another embodiment comprises a kit comprising i) a pharmaceuticallyeffective amount of a compound of Formula (I), (Ia), (Ib), (Ic), (Id),(Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik), and (Il), or apharmaceutically acceptable salt thereof; in a dry powder formulationsuitable for inhalation ii) optionally, one or more pharmaceuticallyacceptable excipients or carriers suitable for inhalation; iii)instructions for administering the compound of group i) and theexcipients or carriers of group ii) to a subject in need thereof; and;iv) a container. In a further embodiment, the kit also comprises a drypowder inhaler suitable for delivering the dry powder formulation to arecipient. The dry powder inhaler may be, in additional embodiments, asingle-dose inhaler or a multi-dose inhaler.

Further embodiments of each of the kits described herein includes thosein which the concentration of the compound of Formula (I), (Ia), (Ib),(Ic), (Id), (Ie), (If), (Ig), (Ih), (II), (Ij), (Ik), and (Il), or apharmaceutically acceptable salt thereof, per dose, is one of theeffective dose ranges described herein, including a) from about 0.1 μgto about 1,000 μg; b) from about 0.5 μg to about 0.5 mg; and c) fromabout 0.5 μg to about 50 μg.

For each of the kits described above there is an additional embodimentin which the diluent is hypertonic saline of the concentrationsdescribed herein. In another embodiment for each kit the diluent ishypotonic saline of the concentrations described herein. In a furtherembodiment for each kit, the diluent is sterile water suitable forinhalation.

The pharmaceutically acceptable excipient(s), diluent(s) or carrier(s)must be acceptable in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Generally, the pharmaceutically acceptable excipient(s),diluent(s) or carrier(s) employed in the pharmaceutical formulation are‘non-toxic’ meaning that it/they is/are deemed safe for consumption inthe amount delivered in the formulation and “inert” meaning that it/theydoes/do not appreciable react with or result in an undesired effect onthe therapeutic activity of the active ingredient(s). Pharmaceuticallyacceptable excipients, diluents and carriers are conventional in the artand may be selected using conventional techniques, based upon thedesired route of administration. See, REMINGTON'S, PHARMACEUTICALSCIENCES, Lippincott Williams & Wilkins; 21^(st) Ed (May 1, 2005).Preferably, the pharmaceutically acceptable excipient(s), diluent(s) orcarrier(s) are Generally Regarded As Safe (GRAS) according to the FDA.

Pharmaceutical compositions according to the invention include thosesuitable for oral administration; parenteral administration, includingsubcutaneous, intradermal, intramuscular, intravenous and intraarticulartopical administration, including topical administration to the skin,eyes, ears, etc; vaginal or rectal administration; and administration tothe respiratory tract, including the nasal cavities and sinuses, oraland extrathoracic airways, and the lungs, including by use of aerosolswhich may be delivered by means of various types of dry powder inhalers,pressurized metered dose inhalers, softmist inhalers, nebulizers, orinsufflators. The most suitable route of administration may depend upon,several factors including the patient and the condition or disorderbeing treated.

The formulations may be presented in unit dosage form or in bulk form asfor example in the case of formulations to be metered by an inhaler andmay be prepared by any of the methods well known in the art of pharmacy.Generally, the methods include the step of bringing the activeingredient into association with the carrier, diluent or excipient andoptionally one or more accessory ingredients. In general theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with one or more liquid carriers,diluents or excipients or finely divided solid carriers, diluents orexcipients, or both, and then, if necessary, shaping the product intothe desired formulation.

In one preferred embodiment, the composition is an inhalablepharmaceutical composition which is suitable for inhalation and deliveryto the endobronchial space. Typically, such composition is in the formof an aerosol comprising particles for delivery using a nebulizer,pressurized metered dose inhaler (MDI), softmist inhaler, or dry powderinhaler (DPI). The aerosol formulation used in the methods of thepresent invention may be a liquid (e.g., solution) suitable foradministration by a nebulizer, softmist inhaler, or MDI, or a dry powdersuitable for administration by an MDI or DPI.

Aerosols used to administer medicaments to the respiratory tract aretypically polydisperse; that is they are comprised of particles of manydifferent sizes. The particle size distribution is typically describedby the Mass Median Aerodynamic Diameter (MMAD) and the GeometricStandard Deviation (GSD). For optimum drug delivery to the endobronchialspace the MMAD is in the range from about 1 to about 10 μm andpreferably from about 1 to about 5 μm, and the GSD is less than 3, andpreferably less than about 2. Aerosols having a MMAD above 10 μm aregenerally too large when inhaled to reach the lungs. Aerosols with a GSDgreater than about 3 are not preferred for lung delivery as they delivera high percentage of the medicament to the oral cavity. To achieve theseparticle sizes in powder formulation, the particles of the activeingredient may be size reduced using conventional techniques such asmicronisation or spray drying. Non-limiting examples of other processesor techniques that can be used to produce respirable particles Includespray drying, precipitation, supercritical fluid, and freeze drying. Thedesired fraction may be separated out by air classification or sieving.In one embodiment, the particles will be crystalline. For liquidformulations, the particle size is determined by the selection of aparticular model of nebulizer, softmist inhaler, or MDI.

Aerosol particle size distributions are determined using devices wellknown in the art. For example a multi-stage Anderson cascade impactor orother suitable method such as those specifically cited within the USPharmacopoeia Chapter 601 as characterizing devices for aerosols emittedfrom metered-dose and dry powder Inhalers.

Dry powder compositions for topical delivery to the lung by inhalationmay be formulated without excipient or carrier and instead Includingonly the active ingredients in a dry powder form having a suitableparticle size for inhalation. Dry powder compositions may also contain amix of the active ingredient and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di- orpoly-saccharides (e.g., lactose or starch). Lactose is typically thepreferred excipient for dry powder formulations. When a solid excipientsuch as lactose is employed, generally the particle size of theexcipient will be much greater than the active ingredient to aid thedispersion of the formulation in the inhaler.

Non-limiting examples of dry powder inhalers include reservoirmulti-dose inhalers, pre-metered multi-dose inhalers, capsule-basedinhalers and single-dose disposable inhalers. A reservoir Inhalercontains a large number of doses (e.g. 60) in one container. Prior toinhalation, the patient actuates the inhaler which causes the inhaler tometer one dose of medicament from the reservoir and prepare it forinhalation. Examples of reservoir DPIs include but are not limited tothe Turbohaler® by AstraZeneca and the ClickHaler® by Vectura.

In a pre-metered multi-dose inhaler, each individual dose has beenmanufactured in a separate container, and actuation of the inhaler priorto inhalation causes a new dose of drug to be released from itscontainer and prepared for inhalation. Examples of multidose DPIinhalers include but are not limited to Diskus® by GSK, Gyrohaler® byVectura, and Prohaler® by Valois. During inhalation, the inspiratoryflow of the patient accelerates the powder out of the device and intothe oral cavity. For a capsule inhaler, the formulation is in a capsuleand stored outside the inhaler. The patient puts a capsule in theinhaler, actuates the inhaler (punctures the capsule), then inhales.Examples include the Rotohaler™ (GlaxoSmithKline), Spinhaler™(Novartis), HandiHaler™ (IB), TurboSpin™ (PH&T). With single-dosedisposable Inhalers, the patient actuates the inhaler to prepare it forinhalation, inhales, then disposes of the inhaler and packaging.Examples include the Twincer™ (U Groningen), OneDose™ (GFE), and MantaInhaler™ (Manta Devices).

Generally, dry powder inhalers utilize turbulent flow characteristics ofthe powder path to cause the excipient-drug aggregates to disperse, andthe particles of active ingredient are deposited in the lungs. However,certain dry powder inhalers utilize a cyclone dispersion chamber toproduce particles of the desired respirable size. In a cyclonedispersion chamber, the drug enters a coin shaped dispersion chambertangentially so that the air path and drug move along the outer circularwall. As the drug formulation moves along this circular wall it bouncesaround and agglomerates are broken apart by impact forces. The air pathspirals towards the center of the chamber exiting vertically. Particlesthat have small enough aerodynamic sizes can follow the air path andexit the chamber. In effect, the dispersion chamber works like a smalljet mill. Depending on the specifics of the formulation, large lactoseparticles may be added to the formulation to aid in the dispersionthrough impact with the API particles.

The Twincer™ single-dose disposable inhaler appears to operate using acoin-shaped cyclone dispersion chamber referred to as an “airclassifier.” See, U.S. Published Patent Application No. 2006/0237010 toRijksuniversiteit Groningen. Papers published by the University ofGroningen, have stated that a 60 mg dose of pure micronized colistinsulfomethate could be effectively delivered as an inhalable dry powderutilizing this technology.

In preferred embodiments, the aerosol formulation is delivered as a drypowder using a dry powder inhaler wherein the particles emitted from theinhaler have an MMAD in the range of about 1 μm) to about 5 μm and a GSDabout less than 2.

Examples of suitable dry powder inhalers and dry powder dispersiondevices for use in the delivery of compounds and compositions accordingto the present invention include but are not limited to those disclosedin U.S. Pat. Nos. 7,520,278; 7,322,354; 7,246,617; 7,231,920; 7,219,665;7,207,330; 6,880,555; 5,522,385; 6,845,772; 6,637,431; 6,329,034;5,458,135; 4,805,811; and U.S. Published Patent Application No.2006/0237010.

In one embodiment, the pharmaceutical formulation according to theinvention is a dry powder for inhalation which is formulated fordelivery by a Diskus®-type device. The Diskus® device comprises anelongate strip formed from a base sheet having a plurality of recessesspaced along its length and a lid sheet hermetically but peelably sealedthereto to define a plurality of containers, each container havingtherein an inhalable formulation containing a predetermined amount ofactive ingredient either alone or in admixture with one or more carriersor excipients (e.g., lactose) and/or other therapeutically activeagents. Preferably, the strip is sufficiently flexible to be wound intoa roll. The lid sheet and base sheet will preferably have leading endportions which are not sealed to one another and at least one of theleading end portions is constructed to be attached to a winding means.Also, preferably the hermetic seal between the base and lid sheetsextends over their whole width. To prepare the dose for inhalation, thelid sheet may preferably be peeled from the base sheet in a longitudinaldirection from a first end of the base sheet.

In one embodiment, the pharmaceutical formulation according to theinvention is a dry powder for inhalation which is formulated fordelivery using a single-dose disposable inhaler, and particularly theTwincer™ inhaler. The Twincer™ Inhaler comprises a foil laminate blisterwith one or more recesses and a lid sheet hermetically but peelablysealed thereto to define a plurality of containers. Each container hastherein an inhalable formulation containing a predetermined amount ofactive ingredient(s) either alone or in admixture with one or morecarriers or excipients (e.g., lactose). The lid sheet will preferablyhave a leading end portion which is constructed to project from the bodyof the inhaler. The patient would operate the device and therebyadminister the aerosol formulation by 1) removing the outer packagingoverwrap, 2) pulling the foil tab to uncover the drug in the blister and3) inhaling the drug from the blister.

In another embodiment, the pharmaceutical formulation according to theinvention is a dry powder for inhalation wherein the dry powder isformulated into microparticles as described in PCT Publication No.WO2009/015286 or WO2007/114881, both to NexBio. Such microparticles aregenerally formed by adding a counter ion to a solution containing acompound of the invention in a solvent, adding an antisolvent to thesolution; and gradually cooling the solution to a temperature belowabout 25° C., to form a composition containing microparticles comprisingthe compound. The microparticles comprising the compound may then beseparated from the solution by any suitable means such as sedimentation,filtration or lyophillization. Suitable counterions, solvents andantisolvents for preparing microparticles of the compounds of theinvention are described in WO2009/015286.

In another embodiment, a pharmaceutical composition according to theinvention is delivered as a dry powder using a metered dose inhaler.Non-limiting examples of metered dose inhalers and devices include thosedisclosed in U.S. Pat. Nos. 5,261,538; 5,544,647; 5,622,163; 4,955,371;3,565,070; 3,361,306 and 6,116,234 and 7,108,159. In a preferredembodiment, a compound of the Invention is delivered as a dry powderusing a metered dose inhaler wherein the emitted particles have an MMADthat is in the range of about 1 μm to about 5 μm and a GSD that is lessthan about 2.

Liquid aerosol formulations for delivery to the endobronchial space orlung by inhalation may for example be formulated as aqueous solutions orsuspensions or as aerosols delivered from pressurized packs, such asmetered dose inhalers, with the use of suitable liquefied propellants,softmist inhalers, or nebulizers. Such aerosol compositions suitable forinhalation can be either a suspension or a solution and generallycontain the active ingredient(s) together with a pharmaceuticallyacceptable carrier or diluent (e.g., water (distilled or sterile),saline, hypertonic saline, or ethanol) and optionally one or more othertherapeutically active agents.

Aerosol compositions for delivery by pressurized metered dose inhalerstypically further comprise a pharmaceutically acceptable propellantExamples of such propellants include fluorocarbon or hydrogen-containingchlorofluorocarbon or mixtures thereof, particularly hydrofluoroalkanes,e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, especially 1,1,1,2-tetrafluoroethane,1,1,1,2,3,3,3,-heptafluoro-n-propane or a mixture thereof. The aerosolcomposition may be excipient free or may optionally contain additionalformulation excipients well known in the art such as surfactants e.g.,oleic acid or lecithin and cosolvents e.g., ethanol. Pressurizedformulations will generally be retained in a canister (e.g., an aluminumcanister) closed with a valve (e.g., a metering valve) and fitted intoan actuator provided with a mouthpiece.

In another embodiment, a pharmaceutical composition according to theInvention is delivered as a liquid using a metered dose inhaler.Non-limiting examples of metered dose inhalers and devices include thosedisclosed in U.S. Pat. Nos. 6,253,762, 6,413,497, 7,601,336, 7,481,995,6,743,413, and 7,105,152. In a preferred embodiment, a compound of theinvention is delivered as a dry powder using a metered dose inhalerwherein the emitted particles have an MMAD that is in the range of about1 μm to about 5 μm and a GSD that is less than about 2.

In one embodiment the aerosol formulation is suitable for aerosolizationby a jet nebulizer, or ultrasonic nebulizer including static andvibrating porous plate nebulizers. Liquid aerosol formulations fornebulization may be generated by solubilizing or reconstituting a solidparticle formulation or may be formulated with an aqueous vehicle withthe addition of agents such as acid or alkali, buffer salts, andisotonicity adjusting agents. They may be sterilized by in-processtechniques such as filtration, or terminal processes such as heating inan autoclave or gamma irradiation. They may also be presented innon-sterile form.

Patients can be sensitive to the pH, osmolality, and ionic content of anebulized solution. Therefore these parameters should be adjusted to becompatible with the active ingredient and tolerable to patients. Themost preferred solution or suspension of active ingredient will containa chloride concentration >30 mM at pH 4.5-7.4, preferably 5.0-5.5, andan osmolality of from about 800-1600 mOsm/kg. The pH of the solution canbe controlled by either titration with common acids (hydrochloric acidor sulfuric acid, for example) or bases (sodium hydroxide, for example)or via the use of buffers. Commonly used buffers include citratebuffers, such as citric acid/sodium citrate buffers, acetate buffers,such as acetic acid/sodium acetate buffers, and phosphate buffers.Buffer strengths can range from 2 mM to 50 mM.

Useful acetate, phosphate, and citrate buffers include sodium acetate,sodium acetate trihydrate, ammonium acetate, potassium acetate, sodiumphosphate, sodium phosphate dibasic, disodium hydrogen phosphate,potassium dihydrogen phosphate, potassium hydrogen phosphate, potassiumphosphate, sodium citrate, and potassium citrate. Other buffers whichmay be utilized include sodium hydroxide, potassium hydroxide, ammoniumhydroxide, aminomethylpropanol, tromethamine, tetrahydroxypropylethylenediamine, citric acid, acetic acid, hydroxytricarboxylic acid ora salt thereof, such as a citrate or sodium citrate salt thereof, lacticacid, and salts of lactic acid including sodium lactate, potassiumlactate, lithium lactate, calcium lactate, magnesium lactate, bariumlactate, aluminum lactate, zinc lactate, silver lactate, copper lactate,iron lactate, manganese lactate, ammonium lactate, monoethanolamine,diethanolamine, triethanolamine, diisopropanolamine, as well ascombinations thereof, and the like.

Such formulations may be administered using commercially availablenebulizers or other atomizer that can break the formulation intoparticles or droplets suitable for deposition in the respiratory tract.Non-limiting examples of nebulizers which may be employed for theaerosol delivery of a composition of the invention include pneumatic jetnebulizers, vented or breath-enhanced jet nebulizers, or ultrasonicnebulizers including static or vibrating porous plate nebulizers.Commercially available nebulizers include the Aeroneb® Go nebulizer(Aerogen) and the eFlow nebulizer (Pad Pharma).

A jet nebulizer utilizes a high velocity stream of air blasting upthrough a column of water to generate droplets. Particles unsuitable forinhalation impact on walls or aerodynamic baffles. A vented or breathenhanced nebulizer works in essentially the same way as a jet nebulizerexcept that Inhaled air passes through the primary droplet generationarea to increase the output rate of the nebulizer while the patientinhales.

In an ultrasonic nebulizer, vibration of a piezoelectric crystal createssurface instabilities in the drug reservoir that cause droplets to beformed. In porous plate nebulizers pressure fields generated by sonicenergy force liquid through the mesh pores where it breaks into dropletsby Rayleigh breakup. The sonic energy may be supplied by a vibratinghorn or plate driven by a piezoelectric crystal, or by the mesh itselfvibrating. Non-limiting examples of atomizers include any single or twinfluid atomizer or nozzle that produces droplets of an appropriate size.A single fluid atomizer works by forcing a liquid through one or moreholes, where the jet of liquid breaks up into droplets. Twin fluidatomizers work by either forcing both a gas and liquid through one ormore holes, or by impinging a jet of liquid against another jet ofeither liquid or gas.

The choice of nebulizer which aerosolizes the aerosol formulation isimportant in the administration of the active ingredient(s). Differentnebulizers have differing efficiencies based their design and operationprinciple and are sensitive to the physical and chemical properties ofthe formulation. For example, two formulations with different surfacetensions may have different particle size distributions. Additionally,formulation properties such as pH, osmolality, and permeant ion contentcan affect tolerability of the medication, so preferred embodimentsconform to certain ranges of these properties.

In a preferred embodiment, the formulation for nebulization is deliveredto the endobronchial space as an aerosol having an MMAD between about 1μm and about 5 μm and a GSD less than 2 using an appropriate nebulizer.To be optimally effective and to avoid upper respiratory and systemicside effects, the aerosol should not have a MMAD greater than about 5 μmand should not have a GSD greater than about 2, if an aerosol has anMMAD larger than about 5 μm or a GSD greater than about 2, a largepercentage of the dose may be deposited in the upper airways decreasingthe amount of drug delivered to the desired site in the lowerrespiratory tract. If the MMAD of the aerosol is smaller than about 1 μmthen a large percentage of the particles may remain suspended in theinhaled air and may then be exhaled during expiration.

The compounds of the invention may also be administered bytransbronchoscopic lavage.

Formulations suitable for oral administration may be presented asdiscrete units such as capsules, cachets or tablets, each containing apredetermined amount of the active ingredient; as a powder or granules;as a solution or suspension in an aqueous liquid or a non-aqueousliquid; or as an oil-in-water liquid emulsion or a water-in-oil liquidemulsion. The active ingredient may also be presented as a sachet,bolus, electuary or paste.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinders, lubricant, Inert diluent, surface active or dispersing agent.Molded tablets may be made by molding in a suitable machine a mixture ofthe powdered compound moistened with an inert liquid diluent. Thetablets may optionally be coated or scored and may be formulated so asto provide slow or controlled release of the active Ingredient therein.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges, comprising the activeingredient in a flavored base such as sucrose and acacia or tragacanth,and pastilles comprising the active ingredient in a base such as gelatinand glycerin or sucrose and acacia.

Formulations for parenteral administration include aqueous andnon-aqueous sterile Injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilized) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Oral fluids such as solutions, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the active ingredient. Syrups can be prepared by dissolvingthe active ingredient in a suitably flavored aqueous solution, whileelixirs are prepared through the use of a pharmaceutically acceptablealcoholic vehicle. Suspensions can be formulated by dispersing theactive ingredient in a pharmaceutically acceptable vehicle. Solubilizersand emulsifiers such as ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be incorporated into oral liquidcompositions.

Liposome delivery systems such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles may also be employed asdelivery means for the compounds of the invention. Liposomes may beformed from a variety of phospholipids such as cholesterol, stearylamineand phosphatidyicholines.

Pharmaceutical compositions for topical administration may be formulatedas ointments, creams, suspensions, lotions, powders, solutions, pastes,gels, sprays, aerosols or oils. Compositions designed for the treatmentof the eyes or other external tissues, for example the mouth and skin,may be applied as a topical ointment or cream. When formulated as anointment, the active ingredient may be employed with either a paraffinicor a water-miscible ointment base. Alternatively, the active ingredientmay be formulated in a cream with an oil-in-water cream base or awater-in-oil base.

Other compositions designed for topical administration to the eyes orears include eye drops and ear drops wherein the active ingredient isdissolved or suspended in a suitable carrier, such as for example anaqueous solvent, including saline.

Compositions designed for nasal administration include aerosols,solutions, suspensions, sprays, mists and drops. Aerosolableformulations for nasal administration may be formulated in much the sameways as aerosolable formulations for inhalation with the condition thatparticles of non-respirable size will be preferred in formulations fornasal administration. Typically, particles of about 5 microns in size,up to the size of visible droplets may be employed. Thus, for nasaladministration, a particle size in the range of 10-500 μm may be used toensure retention in the nasal cavity.

Transdermal patches may also be employed, which are designed to remainin contact with the epidermis of the patient for an extended period oftime and promote the absorption of the active ingredient there through.

Compositions for vaginal or rectal administration include ointments,creams, suppositories and enemas, all of which may be formulated usingconventional techniques.

In another aspect, the invention provides a method of promotinghydration of mucosal surfaces or restoring mucosal defense in a human inneed thereof, comprising administering to the human a pharmaceuticalcomposition comprising a compound of the invention, wherein saidcompound is administered in an effective amount. In one preferredembodiment, the method comprises administering the pharmaceuticalcomposition as an inhalable composition comprising an amount of acompound of the invention that is sufficient to achieve dissolvedconcentration of the compound on the airway surfaces of from about 10⁻⁹,10⁻⁸, or 10⁻⁷ to about 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹ Moles/liter, morepreferably from about 10⁻⁹ to about 10⁻⁴ Moles/liter.

In another aspect, the invention provides a method of treating any oneof: a disease associated with reversible or irreversible airwayobstruction, chronic obstructive pulmonary disease (COPD), asthma,bronchiectasis (including bronchiectasis due to conditions other thancystic fibrosis), acute bronchitis, chronic bronchitis, post-viralcough, cystic fibrosis, emphysema, pneumonia, panbronchiolitis,transplant-associate bronchiolitis, and ventilator-associatedtracheobronchitis or preventing ventilator-associated pneumonia in ahuman in need thereof, comprising administering to the human apharmaceutical composition comprising a compound of the invention,wherein said compound is administered in an effective amount. In onepreferred embodiment, the method comprises administering thepharmaceutical composition as an inhalable composition comprising anamount of a compound of the invention that is sufficient to achievedissolved concentration of the compound on the airway surfaces of fromabout 10⁻⁹, 10⁻⁸, or 10⁻⁷ to about 10⁻⁴, 10⁻³, 10⁻², or 10⁻¹Moles/liter, more preferably from about 10⁻⁹ to about 10⁻⁴ Moles/liter.

In another aspect, the invention provides a method of treating any oneof dry mouth (xerostomia), dry skin, vaginal dryness, sinusitis,rhinosinusitis, or nasal dehydration, including nasal dehydrationbrought on by administering dry oxygen, dry eye or Sjogren's disease,promoting ocular or corneal hydration, treating distal intestinalobstruction syndrome, treating otitis media, primary ciliary diskinesia,distal intestinal obstruction syndrome, esophagitis, constipation, orchronic diverticulitis in a human in need thereof, comprisingadministering to the human a pharmaceutical composition comprising acompound of the invention, wherein said compound is administered in aneffective amount.

Preferred unit dosage formulations for the compounds of the inventionare those containing an effective amount of the active ingredient or anappropriate fraction thereof.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question for example those suitable for oral administration mayinclude flavoring agents.

The compositions of the present invention may be formulated forimmediate, controlled or sustained release as desired for the particularcondition being treated and the desired route of administration. Forexample, a controlled release formulation for oral administration may bedesired for the treatment of constipation in order to maximize deliveryof the active agent to colon. Such formulations and suitable excipientsfor the same are well known in the art of pharmacy. Because the freebase of the compound is generally less soluble in aqueous solutions thanthe salt, compositions comprising a free base of a compound of Formula Imay be employed to provide more sustained release of active agentdelivered by inhalation to the lungs. An active agent present in thelungs in particulate form which has not dissolved into solution is notavailable to induce a physiological response, but serves as a depot ofbioavailable drug which gradually dissolves into solution. As anotherexample, a formulation may employ both a free base and salt form of acompound of the invention to provide both immediate release andsustained release of the active Ingredient for dissolution into themucus secretions of, for example, the nose.

Combinations

The compounds of the invention may be formulated and/or used incombination with other therapeutically active agents. Examples of othertherapeutically active agents which may be formulated or used incombination with the compounds of the invention include but are notlimited to osmolytes, anti-inflammatory agents, anticholinergic agents,β-agonists (including selective β₂-agonists), P2Y2 receptor agonists,peroxisome proliferator-activated receptor (PPAR) delta agonists, otherepithelial sodium channel blockers (ENaC receptor blockers), cysticfibrosis transmembrane conductance regulator (CFTR) modulators, kinaseinhibitors, antiinfective agents, antihistamines, non-antibioticanti-inflammatory macrolides, elastase and protease inhibitors, andmucus or mucin modifying agents, such as surfactants. In addition, forcardiovascular indications, the compounds of the invention may be usedin combination with beta blockers, ACE inhibitors, HMGCoA reductaseinhibitors, calcium channel blockers and other cardiovascular agents.

The present invention thus provides, as another aspect, a compositioncomprising an effective amount of a compound of the invention and one ormore other therapeutically active agents selected from osmolytes,anti-inflammatory agents, anticholinergic agents, β-agonists (includingselective β₂-agonists), P2Y2 receptor agonists, PPAR delta agonists,ENaC receptor blockers, cystic fibrosis transmembrane conductanceregulator (CFTR) modulators, kinase inhibitors, antiinfective agents,antihistamines, non-antibiotic anti-inflammatory macrolides, elastaseand protease inhibitors, and mucus or mucin modifying agents, such assurfactants. The present invention thus provides, as another aspect, acomposition comprising an effective amount of a compound of theinvention and one or more other therapeutically active agents selectedfrom beta blockers, ACE inhibitors, HMGCoA reductase inhibitors, andcalcium channel blockers. Use of the compounds of the invention incombination with one or more other therapeutically active agents(particularly osmolytes) may lower the dose of the compound of theinvention that is required to sufficiently hydrate mucosal surfaces,thereby reducing the potential for undesired side-effects attributableto systemic blocking of sodium channels such as for example in thekidneys.

“Osmolytes” according to the present invention are molecules orcompounds that are osmotically active. “Osmotically active” moleculesand compounds are membrane-impermeable (i.e., essentiallynon-absorbable) on the airway or pulmonary epithelial surface. The terms“airway surface” and “pulmonary surface,” as used herein, includepulmonary airway surfaces such as the bronchi and bronchioles, alveolarsurfaces, and nasal and sinus surfaces. Suitable osmolytes include ionicosmolytes (i.e., salts), and non-Ionic osmolytes (i.e., sugars, sugaralcohols, and organic osmolytes). In general, osmolytes (both ionic andnon-ionic) used in combination with the compounds of the invention arepreferably osmolytes that do not promote, or in fact deter or retardbacterial growth. Osmolytes suitable for use in the present inventionmay be in racemic form or in the form of an enantiomer, diastereomer,tautomer, polymorph or pseudopolymorph.

Examples of ionic osmolytes useful in the present Invention include anysalt of a pharmaceutically acceptable anion and a pharmaceuticallyacceptable cation. Preferably, either (or both) of the anion and cationare osmotically active and not subject to rapid active transport, inrelation to the airway surfaces to which they are administered. Suchcompounds include but are not limited to anions and cations that arecontained in FDA approved commercially marketed salts, see, e.g.,Remington: The Science and Practice of Pharmacy, Vol. II, pg. 1457(19^(th) Ed. 1995), and can be used in any combination as known in theart.

Specific examples of pharmaceutically acceptable osmotically activeanions include but are not limited to, acetate, benzenesulfonate,benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate(camphorsulfonate), carbonate, chloride, citrate, dihydrochloride,edetate, edisylate (1,2-ethanedisulfonate), estolate (lauryl sulfate),esylate (1,2-ethanedisulfonate), fumarate, gluceptate, gluconate,glutamate, glycollylarsanilate (p-glycollamidophenylarsonate),hexylresorcinate, hydrabamine (N,N′-Di(dehydroabietyl)ethylenediamine),hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate,lactate, lactobionate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,nitrite, pamoate (embonate), pantothenate, phosphate or diphosphate,polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate,tannate, tartrate, teoclate (8-chlorotheophyllinate), triethiodide,bicarbonate, etc. Preferred anions include chloride, sulfate, nitrate,gluconate, iodide, bicarbonate, bromide, and phosphate.

Specific examples of pharmaceutically acceptable osmotically activecations include but are not limited to, organic cations such asbenzathine (N,N′-dibenzylethylenediamine), chloroprocaine, choline,diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine),procaine, D-lysine. L-lysine, D-arginine, L-arginine, triethylammonium,N-methyl D-glycerol, and the like; and metallic cations such asaluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron,ammonium, and the like. Preferred organic cations include 3-carbon,4-carbon, 5-carbon and 6-carbon organic cations. Preferred cationsinclude sodium, potassium, choline, lithium, meglumine, D-lysine,ammonium, magnesium, and calcium.

Specific examples of ionic osmolytes that may be used in combinationwith a compound of the invention include but are not limited to, sodiumchloride (particularly hypertonic saline), potassium chloride, cholinechloride, choline iodide, lithium chloride, meglumine chloride, L-lysinechloride, D-lysine chloride, ammonium chloride, potassium sulfate,potassium nitrate, potassium gluconate, potassium iodide, ferricchloride, ferrous chloride, potassium bromide, and combinations of anytwo or more of the foregoing. In one embodiment, the present inventionprovides a combination of a compound of the invention and two differentosmotically active salts. When different salts are used, one of theanion or cation may be the same among the differing salts. Hypertonicsaline is a preferred ionic osmolyte for use in combination with thecompounds of the invention.

Non-ionic osmolytes include sugars, sugar-alcohols, and organicosmolytes. Sugars and sugar-alcohols useful as osmolytes in the presentinvention include but are not limited to 3-carbon sugars (e.g.,glycerol, dihydroxyacetone); 4-carbon sugars (e.g., both the D and Lforms of erythrose, threose, and erythrulose); 5-carbon sugars (e.g.,both the D and L forms of ribose, arabinose, xylose, lyxose, psicose,fructose, sorbose, and tagatose); and 6-carbon sugars (e.g., both the Dand L forms of altose, allose, glucose, mannose, gulose, idose,galactose, and talose, and the D and L forms of allo-heptulose,allo-hepulose, gluco-heptulose, manno-heptulose, gulo-heptulose,ido-heptulose, galacto-heptulose, talo-heptulose). Additional sugarsuseful in the practice of the present invention include raffinose,raffinose series oligosaccharides, and stachyose. Both the D and L formsof the reduced form of each sugar/sugar alcohol are also suitable forthe present invention. For example, glucose, when reduced, becomessorbitol; an osmolyte within the scope of the invention. Accordingly,sorbitol and other reduced forms of sugar/sugar alcohols (e.g.,mannitol, dulcitol, arabitol) are suitable osmolytes for use in thepresent invention. Mannitol is a preferred non-Ionic osmolyte for use incombination with the compounds of the invention.

“Organic osmolytes” is generally used to refer to molecules that controlintracellular osmolality in the kidney. See e.g., J. S. Handler et al.,Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol.268, F983-F996 (1995). Organic osmolytes include but are not limited tothree major classes of compounds: polyols (polyhydric alcohols),methylamines, and amino acids. Suitable polyol organic osmolytes includebut are not limited to, inositol, myo-inositol, and sorbitol. Suitablemethylamine organic osmolytes include but are not limited to, choline,betaine, carnitine (L-, D- and DL forms), phosphorylcholine,lyso-phosphorylcholine, glycerophosphorylcholine, creatine, and creatinephosphate. Suitable amino acid organic osmolytes include but are notlimited to, the D- and L-forms of glycine, alanine, glutamine,glutamate, aspartate, proline and taurine. Additional organic osmolytessuitable for use in the present invention include tihulose andsarcosine. Mammalian organic osmolytes are preferred, with human organicosmolytes being most preferred. However, certain organic osmolytes areof bacterial, yeast, and marine animal origin, and these compounds mayalso be employed in the present invention.

Osmolyte precursors may be used in combination with the compounds of theinvention An “osmolyte precursor” as used herein refers to a compoundwhich is converted into an osmolyte by a metabolic step, eithercatabolic or anabolic. Examples of osmolyte precursors include but arenot limited to, glucose, glucose polymers, glycerol, choline,phosphatidylcholine, lyso-phosphatidylcholine and inorganic phosphates,which are precursors of polyols and methylamines. Precursors of aminoacid osmolytes include proteins, peptides, and polyamino adds, which arehydrolyzed to yield osmolyte amino acids, and metabolic precursors whichcan be converted into osmolyte amino acids by a metabolic step such astransamination. For example, a precursor of the amino acid glutamine ispoly-L-glutamine, and a precursor of glutamate is poly-L-glutamic acid.

Chemically modified osmolytes or osmolyte precursors may also beemployed. Such chemical modifications involve linking the osmolyte (orprecursor) to an additional chemical group which alters or enhances theeffect of the osmolyte or osmolyte precursor (e.g., inhibits degradationof the osmolyte molecule). Such chemical modifications have beenutilized with drugs or prodrugs and are known in the art. (See, forexample, U.S. Pat. Nos. 4,479,932 and 4,540,564; Shek, E. et al., J.Med. Chem. 19:113-117 (1976); Bodor, N. et al., J. Pharm. Sci.67:1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26:313-318 (1983);Bodor, N. et al., J. Pharm. Sci. 75:29-35 (1986).

Preferred osmolytes for use in combination with the compounds of theinvention include sodium chloride, particular hypertonic saline, andmannitol.

For the formulation of 7% and >7% hypertonic saline, formulationscontaining bicarbonate anions may be particularly useful, especially forrespiratory disorders with cystic fibrosis transmembrane conductanceregulator (CFTR) dysfunction such as CF or COPD. Recent findingsindicate that, although the relative ratio of HCO₃ ⁻ conductance/Cl⁻conductance is between 0.1 and 0.2 for single CFTR channels activatedwith cAMP and ATP, the ratio in the sweat duct can range from virtually0 to almost 1.0, depending on conditions of stimulation. That is,combining cAMP+cGMP+α-ketoglutarate can yield CFTR HCO₃ ⁻ conductancealmost equal to that of Cl⁻ conductance (Quiton et al. Physiology, Vol.22, No. 3, 212-225, June 2007). Furthermore, formulations of 7% and >7%hypertonic saline containing bicarbonate anions may be particularlyuseful due to better control of the pH in the airway surface liquid.First, it has shown that that airway acidification occurs in CF (Tate etal. 2002) and that absent CFTR-dependent bicarbonate secretion can leadto an impaired capacity to respond to airway conditions associated withacidification of airway surface liquid layer (Coakley et al. 2003).Second, addition of HS solution without bicarbonate to the surface ofthe lung may further dilute the bicarbonate concentrations, andpotentially reduce the pH or the ability to respond to airwayacidification within the airway surface liquid layer. Therefore additionof bicarbonate anions to HS may help maintain or improve the pH ofairway surface liquid layer in CF patients.

Due to this evidence, inclusion of bicarbonate anion in the formulationof 7% or >7% hypertonic saline administered by the method of thisinvention would be particularly useful. Formulations containing up to 30to 200 mM concentrations of bicarbonate anions are of particularinterest for 7% or >7% HS solutions.

Hypertonic saline is understood to have a salt concentration greaterthan that of normal saline (NS), i.e. greater than 9 g/L or 0.9% w/v,and hypotonic saline has a salt concentration less than that of normalsaline, such as from about 1 g or L/0.1% w/v to about 8 g/L or 0.8% w/v.Hypertonic saline solutions useful in the formulations and methods oftreatment herein may have a salt concentration from about 1% to about23.4% (w/v). In one embodiment the hypertonic saline solution has a saltconcentration from about 60 g/L (6% w/v) to about 100 g/L (10% w/v). Inanother embodiment, the saline solution has a salt concentration fromabout 70 g/L (7% w/v) to about 100 g/L (10% w/v). In furtherembodiments, the saline solution has salt concentrations of a) fromabout 0.5 g/L (0.05% w/v) to about 70 g/L (7% w/v); b) from about 1 g/L(0.1% w/v) to about 60 g/L (6% w/v); c) from about 1 g/L (0.1% w/v) toabout 50 g/L (5% w/v); d) from about 1 g/L (0.1% w/v) to about 40 g/L(4% w/v); e) from about 1 g/L (0.1% w/v) to about 30 g/L (3% w/v); andf) from about 1 g/L (0.1% w/v) to about 20 g/L (2% w/v).

Specific concentrations of saline solutions useful in the formulationsand methods of treatment herein include, independently, those havingsalt concentrations of 1 g/L (0.1% w/v), 2 g/L (0.2% w/v), 3 g/L (0.3%w/v), 4 g/L (0.4% w/v), 5 g/L (0.5% w/v), 6 g/L (0.6% w/v), 7 g/L (0.7%w/v), 8 g/L (0.8% w/v), 9 g/L (0.9% w/v), 10 g/L (1% w/v), 20 g/L (2%w/v), 30 g/L (3% w/v), 40 g/L (4% w/v), 50 g/L (5% w/v), 60 g/L (6%w/v), 70 g/L (7% w/v), 80 g/L (8% w/v), 90 g/L (9% w/v), 100 g/L (10%w/v), 110 g/L (11% w/v), 120 g/L (12% w/v), 130 g/L (13% w/v), 140 g/L(14% w/v), 150 g/L (15% w/v), 160 g/L (16% w/v), 170 g/L (17% w/v), 180g/L (18% w/v), 190 g/L (19% w/v), 200 g/L (20% w/v), 210 g/L (21% w/v),220 g/L (22% w/v), and 230 g/L (23% w/v). Saline concentrations betweeneach of these listed concentrations/percentages may also be used, suchas saline of 1.7 g/L (0.17% w/v), 1.25 g/L (1.25% w/v), 1.5 g/L (1.5%w/v), 25 g/L (2.5% w/v), 28 g/L (2.8% w/v), 35 g/L (3.5% w/v), 45 g/L(4.5% w/v), and 75 g/L (7.5% w/v).

Specific useful concentration of hypotonic saline solutions includethose from about 0.12 g/L (0.012% w/v) to about 8.5 g/L (0.85% w/v). Anyconcentration within this range may be used, such as, on a w/v basis,0.05%, 0.1%, 0.15%, 0.2%, 0.225% (¼ NS), 0.25%, 0.3% (⅓ NS), 0.35%,0.4%, 0.45% (½ NS), 0.5%, 0.55%, 0.6% (⅔ NS), 0.65%, 0.675% (¾ NS),0.7%, 0.75%, and 0.8%.

Each of the ranges and specific concentrations of saline describedherein may be used with the formulations, methods of treatment,regimens, and kits described herein.

Also intended within the scope of this invention are chemically modifiedosmolytes or osmolyte precursors. Such chemical modifications involvelinking to the osmolyte (or precursor) an additional chemical groupwhich alters or enhances the effect of the osmolyte or osmolyteprecursor (e.g., inhibits degradation of the osmolyte molecule). Suchchemical modifications have been utilized with drugs or prodrugs and areknown in the art. (See, for example, U.S. Pat. Nos. 4,479,932 and4,540,564; Shek, E. et al., J. Med. Chem. 19:113-117 (1976); Bodor, N.et al., J. Pharm. Sci. 67:1045-1050 (1978); Bodor, N. et al., J. Med.Chem. 26:313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75:29-35(1986), each incorporated herein by reference.

Suitable anti-inflammatory agents for use in combination with thecompounds of the invention include corticosteroids and non-steroidalanti-inflammatory drugs (NSAIDs), particularly phosphodiesterase (PDE)inhibitors. Examples of corticosteroids for use in the present inventioninclude oral or inhaled corticosteroids or prodrugs thereof. Specificexamples include but are not limited to ciclesonide,desisobutyryl-ciclesonide, budesonide, flunisolide, mometasone andesters thereof (e.g., mometasone furoate), fluticasone propionate,fluticasone furoate, beclomethasone, methyl prednisolone, prednisolone,dexamethasone,6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxo-androsta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester,6α,9α-difluoro-11-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene-17β-carbothioicacid S-(2-oxo-tetrahydro-furan-3S-yl) ester, beclomethasone esters(e.g., the 17-propionate ester or the 17,21-dipropionate ester,fluoromethyl ester, triamcinolone acetonide, rofleponide, or anycombination or subset thereof. Preferred corticosteroids for formulationor use in combination with the compounds of the invention are selectedfrom ciclesonide, desisobutyryl-ciclesonide, budesonide, mometasone,fluticasone propionate, and fluticasone furoate, or any combination orsubset thereof.

NSAIDs for use in the present invention include but are not limited tosodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE)inhibitors (e.g., theophylline, aminophylline, PDE4 inhibitors, mixedPDE3/PDE4 inhibitors or mixed PDE4/PDE7 inhibitors), leukotrieneantagonists, inhibitors of leukotriene synthesis (e.g., 5 LO and FLAPinhibitors), nitric oxide synthase (iNOS) Inhibitors, proteaseInhibitors (e.g., tryptase inhibitors, neutrophil elastase inhibitors,and metalloprotease inhibitors) β2-integrin antagonists and adenosinereceptor agonists or antagonists (e.g., adenosine 2a agonists), cytokineantagonists (e.g., chemokine antagonists) or inhibitors of cytokinesynthesis (e.g., prostaglandin D2 (CRTh2) receptor antagonists).Examples of leukotriene modifiers suitable for administration by themethod of this invention include montelukast, zileuton and zafirlukast.

The PDE4 Inhibitor, mixed PDE3/PDE4 inhibitor or mixed PDE4/PDE7inhibitor may be any compound that is known to inhibit the PDE4 enzymeor which is discovered to act as a PDE4 inhibitor, and which areselective PDE4 inhibitors (I.e., compounds which do not appreciablyinhibit other members of the PDE family). Examples of specific PDE4inhibitors for formulation and use in combination with the compounds ofthe present invention Include but are not limited to roflumilast,pumafentrine, arofylline, cilomilast, tofimilast, oglemilast,tolafentrine, pidamilast, ibudilast, apremilast,2-[4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthaleny)]-2-pyridinyl]-4-(3-pyridinyl)-1(2H)-phthalazinone(T2585),N-(3,5-dichloro-4-pyridinyl)-1-[(4-fluorophenyl)methyl]-5-hydroxy-α-oxo-1H-indole-3-acetamide(AWD-12-281,4-[(2R)-2-[3-(cyclopentyloxy)-4-methoxyphenyl]-2-phenylethyl]-pyridine(CDP-840),2-[4-[[[[2-(1,3-benzodioxol-5-yloxy)-3-pyridinyl]carbonyl]amino]methyl]-3-fluorophenoxy]-(2R)-propanoicacid (CP-671305),N-(4,6-dimethyl-2-pyrimidinyl)-4-[4,5,6,7-tetrahydro-2-(4-methoxy-3-methylphenyl)-5-(4-methyl-1-piperazinyl)-1H-indol-1-yl]-benzenesulfonamide,(2E)-2-butenedioate (YM-393059),9-[(2-fluorophenyl)methyl]-N-methyl-2-(trifluoromethyl)-9H-purin-6-amine(NCS-613), N-(2,5-dichloro-3-pyridinyl)-8-methoxy-5-quinolinecarboxamide(D-4418),N-[(3R)-9-amino-3,4,6,7-tetrahydro-4-oxo-1-phenylpyrolo[3,2,1-][1,4]benzodiazepin-3-yl]-3H-purin-6-amine(PD-168787),3-[[3-(cydopentyloxy)-4-methoxyphenyl]methyl]-N-ethyl-8-(1-methylethyl)-3H-purin-6-aminehydrochloride (V-11294A).N-(3,5-dichloro-1-oxido-4-pyridinyl)-8-methoxy-2-(trifluoromethyl)-5-quinolinecarboxamide(Sch351591),5-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-[(3-methylphenyl)methyl]-(3S,5S)-2-piperidinone(HT-0712),5-(2-((1R,4R)-4-amino-1-(3-(cyclopentyloxy)-4-methyoxyphenyl)cyclohexyl)ethynyl)-pyrimidine-2-amine,cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol], and4-[6,7-diethoxy-2,3-bis(hydroxymethyl)-1-naphthalenyl]-1-(2-methoxyethyl)-2(1H)-pyridinone(T-440), and any combination or subset thereof.

Leukotriene antagonists and inhibitors of leukotriene synthesis includezafirlukast, montelukast sodium, zileuton, and pranlukast.

Anticholinergic agents for formulation or use in combination with thecompounds of the invention include but are not limited to muscarinicreceptor antagonists, particularly including pan antagonists andantagonists of the M₃ receptors. Exemplary compounds include thealkaloids of the belladonna plants, such as atropine, scopolamine,homatropine, hyoscyamine, and the various forms including salts thereof(e.g., anhydrous atropine, atropine sulfate, atropine oxide or HCl,methylatropine nitrate, homatropine hydrobromide, homatropine methylbromide, hyoscyamine hydrobromide, hyoscyamine sulfate, scopolaminehydrobromide, scopolamine methyl bromide), or any combination or subsetthereof.

Additional anticholinergics for formulation and use in combination withthe methantheline, propantheline bromide, anisotropine methyl bromide orValpin 50, aclidinium bromide, glycopyrrolate (Robinul), isopropamideIodide, mepenzolate bromide, tridihexethyl chloride, hexocycliummethylsulfate, cyclopentolate HCl, tropicamide, trihexyphenidyl CCI,pirenzepine, telenzepine, and methoctramine, or any combination orsubset thereof.

Preferred anticholinergics for formulation and use in combination withthe compounds of the invention include ipratropium (bromide), oxitropium(bromide) and tiotropium (bromide), or any combination or subsetthereof.

Examples of β-agonists for formulation and use in combination with thecompounds of the invention Include but are not limited to salmeterol,R-salmeterol, and xinafoate salts thereof, albuterol or R-albuterol(free base or sulfate), levalbuterol, salbutamol, formoterol (fumarate),fenoterol, procaterol, pirbuterol, metaprterenol, terbutaline and saltsthereof, and any combination or subset thereof.

P2Y2 receptor agonists for formulation and use in combination with thecompounds of the invention may be employed in an amount effective tostimulate chloride and water secretion by airway surfaces, particularlynasal airway surfaces. Suitable P2Y2 receptor agonists are known in theart and are described for example, in columns 9-10 of U.S. Pat. No.6,264,975, and also U.S. Pat. Nos. 5,656,256 and 5,292,498.

P2Y₂ agonists that can be administered by the methods of this inventioninclude P2Y₂ receptor agonists such as ATP, UTP, UTP-.gamma.-S anddinucleotide P2Y₂ receptor agonists (e.g. denufosol or diquafosol) or apharmaceutically acceptable salt thereof. The P2Y₂ receptor agonist istypically included in an amount effective to stimulate chloride andwater secretion by airway surfaces, particularly nasal airway surfaces.Suitable P2Y₂ receptor agonists are described in, but are not limitedto, U.S. Pat. Nos. 6,264,975, 5,656,256, 5,292,498, 6,348,589,6,818,629, 6,977,246, 7,223,744, 7,531,525 and U.S. Pat.AP.2009/0306009each of which is incorporated herein by reference.

Combination therapies and formulations herein can include adenosine 2b(A2b) agonists, also, including BAY 60-6583, NECA(N-ethylcarboxamidoadenosine), (S)-PHPNECA, LUF-5835 and LUF-5845. A2bagonists that may be used are described by Volpini et al., Journal ofMedicinal Chemistry 45 (15): 3271-9 (2002); Volpini et al., CurrentPharmaceutical Design 8 (26): 2285-08 (2002); Baraldi et al., Journal ofMedicinal Chemistry 47 (6): Cacciari et al., 1434-47 (2004); MiniReviews in Medicinal Chemistry 5 (12): 1053-60 (December 2005); Baraldiet al., Current Medicinal Chemistry 13 (28): 3467-82 (2006); Beukers etal., Medicinal Research Reviews 28 (5): 667-98 (September 2006); Elzeinet al., Bioorganic & Medicinal Chemistry Letters 16 (2): 302-6 (January2006); Carotti, et al., Journal of Medicinal Chemistry 49 (1): 282-99(January 2006); Tabrizi et al., Bioorganic & Medicinal Chemistry 16 (5):2419-30 (March 2008); and Stefanachi, et al., Bioorganic & MedicinalChemistry 16 (6): 2852-69 (March 2008).

Examples of other ENaC receptor blockers for formulation and use incombination with the compounds of the invention include but are notlimited to amiloride and derivatives thereof such as those compoundsdescribed in U.S. Pat. No. 6,858,615, and PCT Publication Nos.WO2003/070182, WO2004/073629, WO2005/018644, WO2006/022935,WO2007/018640, and WO2007/146869, all to Parion Sciences, Inc.

Small molecule ENaC blockers are capable of directly preventing sodiumtransport through the ENaC channel pore. ENaC blocker that can beadministered in the combinations herein Include, but are not limited to,amiloride, benzamil, phenamil, and amiloride analogues as exemplified byU.S. Pat. Nos. 6,858,614, 6,858,615, 6,903,105, 6,995,160, 7,026,325,7,030,117, 7,064,129, 7,186,833, 7,189,719, 7,192,958, 7,192,959,7,241,766, 7,247,636, 7,247,637, 7,317,013, 7,332,496, 7,345,044,7,388,447, 7,368,450, 7,368,451, 7,375,107, 7,399,766, 7,410,968,7,820,678, 7,842,697, 7,868,010, 7,875,619.

ENaC proteolysis is well described to increase sodium transport throughENaC. Protease inhibitor block the activity of endogenous airwayproteases, thereby preventing ENaC cleavage and activation. Proteasethat cleave ENaC include furin, meprin, matriptase, trypsin, channelassociated proteases (CAPs), and neutrophil elastases. Proteaseinhibitors that can inhibit the proteolytic activity of these proteasesthat can be administered in the combinations herein include, but are notlimited to, camostat, prostasin, furin, aprotinin, leupeptin, andtrypsin inhibitors.

Combinations herein may include one or more suitable nucleic acid (orpolynucleic acid), including but not limited to antisenseoligonucleotide, siRNA, miRNA, miRNA mimic, antagomir, ribozyme,aptamer, and decoy oligonucleotide nucleic acids. See, e.g., US PatentApplication Publication No. 20100316628. In general, such nucleic acidsmay be from 17 or 19 nucleotides in length, up to 23, 25 or 27nucleotides in length, or more. Examples include, but are not limitedto, those described in U.S. Pat. No. 7,517,865 and US PatentApplications Nos. 20100215588; 20100316628; 20110008366; and20110104255. In general, the siRNAs are from 17 or 19 nucleotides inlength, up to 23, 25 or 27 nucleotides in length, or more.

CFTR activity modulating compounds that can be administered in thecombinations of this invention include, but are not limited to,compounds described in US 2009/0246137 A1, US 2009/0253736 A1, US2010/0227888 A1, U.S. Pat. No. 7,645,789, US 2009/0246820 A1, US2009/0221597 A1, US 2010/0184739 A1, US 2010/0130547 A1, US 2010/0168094A1 and issued patent: U.S. Pat. Nos. 7,553,855; 7,772,259 B2, 7,405,233B2, US 2009/0203752, U.S. Pat. No. 7,499,570.

Mucus or mucin modifying agents useful in the combinations and methodsherein include reducing agents, surfactants and detergents,expectorants, and deoxyribonuclease agents.

Mucin proteins are organized into high molecular weight polymers via theformation of covalent (disulfide) and non-covalent bonds. Disruption ofthe covalent bonds with reducing agents is a well-established method toreduce the viscoelastic properties of mucus in vitro and is predicted tominimize mucus adhesiveness and improve clearance in vivo. Reducingagents are well known to decrease mucus viscosity in vitro and commonlyused as an aid to processing sputum samples⁸. Examples of reducingagents include sulfide containing molecules or phosphines capable ofreducing protein di-sulfide bonds including, but not limited to,N-acetyl cysteine, N-acystelyn, carbocysteine, glutathione,dithiothreitol, thioredoxin containing proteins, and tris(2-carboxyethyl) phosphine.

N-acetyl cysteine (NAC) is approved for use in conjunction with chestphysiotherapy to loosen viscid or thickened airway mucus⁽¹²⁾. Clinicalstudies evaluating the effects of oral or inhaled NAC in CF and COPDhave reported improvements in the rheologic properties of mucus andtrends toward improvements in lung function and decreases in pulmonaryexacerbations⁹. However, the preponderance of clinical data suggeststhat NAC is at best a marginally effective therapeutic agent fortreating airway mucus obstruction when administered orally or byinhalation. A recent Cochrane review of the existing clinical literatureon the use of NAC found no evidence to support the efficacy of NAC forCF¹⁰. The marginal clinical benefit of NAC reflects:

NAC is a relative inefficient reducing agent which is only partiallyactive on the airway surface. Very high concentrations of NAC (200 mM or3.26%) are required to fully reduce Muc5B, a major gel-forming airwaymucin, in vitro. Furthermore, in the pH environment of the airwaysurface (measured in the range of pH 6.0 to 7.2 in CF and COPDairways)¹¹, NAC exists only partially in its reactive state as anegatively charge thiolate. Thus, in the clinic, NAC is administered atvery high concentrations. However, it is predicted that current aerosoldevices will not be able to achieve therapeutic concentrations of even a20% Mucomyst solution on distal airway surfaces within the relativelyshort time domains (7.5-15 minutes) typically used.

In non-clinical studies, ¹⁴C-labeled NAC, administered by inhalation,exhibits rapid elimination from the lungs with a half-life ranging from6 to 36 minutes¹²

NAC is administered as a highly concentrated, hypertonic inhalationsolution (20% or 1.22 molar) and has been reported to causebronchoconstriction and cough. In many cases, it is recommended that NACbe administered with a bronchodilator to improve the tolerability ofthis agent.

Thus, reducing agents such as NAC are not well suited for bolus aerosoladministration. However, it is anticipated that delivery of reducingagents by pulmonary aerosol infusion would increase the effectiveness,while allowing for a decrease in the concentration of reducing agent inthe inhalation solution (predicted to increase tolerability).

Surfactants and detergents are spreading agents shown to decrease mucusviscoelasticity, improving mucus clearability. Examples of surfactantsinclude dipalmitoylphosphatidylcholine (DPPC), PF, palmitic acid,palmitoyl-oleoylphosphatidylglycerol, surfactant-associated proteins(e.g. SP-A, B, or C), or may be animal derived (e.g. from cow or calflung lavage or extracted from minced pig lung) or combinations thereof.See, e.g., U.S. Pat. Nos. 7,897,577; 5,876,970; 5,614,216; 5,100,806;and 4,312,860. Examples of surfactant products include Exosurf® Neonatal(colfosceril palmitate), Pumactant® (DPPC and egg phosphatidylglycerol),KL-4 surfactant, Venticute® (Iusulptide, rSP-C surfactant), Alveofact®(bovactant), Curosurf® (poractant alfa), Infasurf® (calfactant),Newfacten® (modified bovine surfactant), Surface®, Natsurf™ (nonionicalcohol ethoxylate surfactant) and Survanta® (beractant). Examples ofdetergents include, but are not limited to, Tween-80 and triton-X 100.

Any suitable expectorant can be used, including but not limited togualfenesin (see, e.g., U.S. Pat. No. 7,345,051). Any suitabledeoxyribonuclease can be used, including but not limited to DomaseAlpha. (see, e.g., U.S. Pat. No. 7,482,024). Examples of kinaseinhibitors include inhibitors of NFkB, PI3K (phosphatidylinositol3-kinase), p38-MAP kinase and Rho kinase.

Antiinfective agents for formulation and use in combination with thecompounds of the invention include antivirals and antibiotics. Examplesof suitable antivirals Include Tamiflu® (oseltamivir) and Relenza®(zanamivir). Examples of suitable antibiotics include but are notlimited to aztreonam (arginine or lysine), fosfomycin, andaminoglycosides such as tobramycin, or any combination or subsetthereof. Additional antiinfective agents that may be used herein includeaminoglycosides, Daptomycin, Fluoroquinolones. Ketolides, Carbapenems,Cephalosporins, Erythromycin, Linezolid, Penicillins, Azithromycidn,Clindamycin, Oxazolidinones, Tetracyclines, and Vancomycin.

Examples of useful carbapenam antibiotics are impenam, panipenam,meropenam, biapenam, MK-826 (L-749,345), DA-1131, ER-35786, lenapenam,S-4661, CS-834 (prodrug of R-95867), KR-21056 (prodrug of KR-21012),L-084 (prodrug of LJC 11036) and Ceftolozane (CXA-101).

Antihistamines (i.e., H1-receptor antagonists) for formulation and usein combination with the compounds of the invention include but are notlimited to: ethanolamines such as diphenhydramine HCl, carbinoxaminemaleate, doxylamine, clemastine fumarate, diphenylhydramine HCl anddimenhydrinate; ethylenediamines such as pyrilamine maleate(metpyramine), tripelennamine HCl, tripelennamine citrate, andantazoline; alkylamines such as pheniramine, chloropheniramine,bromopheniramine, dexchlorpheniramine, triprolidine and acrivastine;pyridines such as methapyrilene, piperazines such as hydroxyzine HCl,hydroxyzine pamoate, cyclizine HCl, cyclizine lactate, meclizine HCl andcetirizine HCl; piperidines such as astemisole, levocabastine HCl,loratadine, descarboethoxyloratadine, terfenadine, and fexofenadine HCl;tri- and tetracyclics such as promethazine, chlorpromethazinetrimeprazine and azatadine; and azelastine HCl, or any combination orsubset thereof.

Examples of other classes of therapeutic agents suitable for use in thecombinations and methods herein Include antivirals such as ribavirin,anti-fungal agents such as amphotencin, intraconazol and voriconazol,anti-rejection drugs such as cyclosporine, tacrolimus and sirolimus,bronchodilators including but not limited to anticholinergic agents suchas atrovent, siRNAs, gene therapy vectors, aptamers, endothelin-receptorantagonists, alpha-1-antitrypsin and prostacyclins.

In the above-described methods of treatment and uses, a compound of theinvention may be employed alone, or in combination with one or moreother therapeutically active agents. Typically, any therapeuticallyactive agent that has a therapeutic effect in the disease or conditionbeing treated with the compound of the invention may be utilized incombination with the compounds of the invention, provided that theparticular therapeutically active agent is compatible with therapyemploying a compound of the invention. Typical therapeutically activeagents which are suitable for use in combination with the compounds ofthe invention include agents described above.

In one preferred embodiment, the compounds of the invention are used incombination with one or more osmolytes, particularly hypertonic salineor mannitol.

In another aspect, the invention provides methods for treatment and usesas described above, which comprise administering an effective amount ofa compound of the invention and at least one other therapeuticallyactive agent. The compounds of the invention and at least one additionaltherapeutically active agent may be employed in combinationconcomitantly or sequentially in any therapeutically appropriatecombination. The administration of a compound of the invention with oneor more other therapeutically active agents may be by administrationconcomitantly in 1) a unitary pharmaceutical composition, such as thecompositions described above, or 2) separate pharmaceutical compositionseach including one or more of the component active ingredients. Thecomponents of the combination may be administered separately in asequential manner wherein the compound of the invention is administeredfirst and the other therapeutically active agent is administered secondor vice versa.

In the embodiments wherein the compound of the invention is administeredin combination with one or more osmolytes, the administration of eachcomponent is preferably concomitant, and may be in a unitary compositionor separate compositions. In one embodiment, the compound of theinvention and one or more osmolytes are administered concomitantly bytransbronchoscopic lavage. In another embodiment, the compound of theinvention and one or more osmolytes are administered concomitantly byinhalation.

When a compound of the invention is used in combination with anothertherapeutically active agent, the dose of each compound may differ fromthat when the compound of the invention is used alone. Appropriate doseswill be readily determined by one of ordinary skill in the art. Theappropriate dose of the compound of the invention, the othertherapeutically active agent(s) and the relative timings ofadministration will be selected in order to achieve the desired combinedtherapeutic effect, and are within the expertise and discretion of theattendant physician, clinician or veterinarian.

Experimental Procedures

The present invention also provides processes for preparing thecompounds of the invention and to the synthetic intermediates useful insuch processes, as described in detail below.

Certain abbreviations and acronyms are used in describing the syntheticprocesses and experimental details. Although most of these would beunderstood by one skilled in the art, the following table contains alist of many of these abbreviations and acronyms.

Abbreviation Meaning AcOH Acetic Add AlBN Azobisisobutyrolnitrile DIADDiisopropyl azidocarboxylate DIPEA N,N-Diisopropylethylamine DCEdichloroethnne DCM dichloromethane AcOH Acetic Acid DMFdimethylformamide Et Ethyl EtOAr or EA ethyl acetate EtOH Ethanol ESIelectrospray ionization HATU2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uroniumhexafluorophosphate HPLC High performance liquid chromatography iPrOHIsopropyl alcohol i.t. or IT intratracheal Me Methyl MeOH methanol m/zor m/e mass to charge ratio MH⁺ mass plus 1 MH⁻ mass minus 1 MIC minimalinhibitory concentration MS or ms mass spectrum rt or r.t. roomtemperature R_(f) Retardation factor t-Bu tert-butyl THF tetrahydrofuranTLC or tlc thin layer chromatography δ parts per million down field fromtetramethylsilane Cbz Benzyloxycarbonyl, i.e. —(CO)O-benzyl AUC Areaunder the curve or peak MTBE Methyl tertiary butyl ether t_(R) Retentiontime GC-MS Gas chromatography-mass spectrometry wt % Percent by weight hHours min Minutes AcOH Acetic Mid MHz megahertz TFA Trifluoroacetic acidUV Ultraviolet Boc tert-butyloxycarbonyl DIAD Diiscpropylazodicarboxylate AcOH Acetic Acid DIPEA N,N-Diisopropylethylamine orHünig's base Ph₃P TriphenylphosineThe compounds of Formula I may be synthesized using techniques known inthe art. A representative synthetic procedure is Illustrated in Scheme 1below.

These procedures are described in, for example, E. J. Cragoe, “TheSynthesis of Amiloride and Its Analogs” (Chap 3) in Amiloride and ItsAnalogs, pp. 25-36. Other processes for preparing amiloride analogs aredescribed in, for example, U.S. Pat. No. 3,318,813, to Cragoe,particularly at methods A, B, C, and D of the '813 patent. Still otherprocesses which may be adapted for the preparation of the compounds ofthe invention are described in PCT Publication Nos. WO2003/07182,WO2005/108644, WO2005/022935, U.S. Pat. Nos. 7,064,129, 6,858,615,6,903,105, WO 2004/073629, WO 2007/146869, and WO 2007/018640, allassigned to Parion Sciences. Inc.

Preparation of methylN′-3,5-diamino-6-chloropyrazine-2-carbonylcarbamimido thioate (2) can beseen in WO 2009/074575.

Generally, the compounds of the invention may be conveniently preparedby treating a compound of Formula 2 with an amine of Formula 3. Morespecifically, compounds of Formula 2 are treated with the amine ofFormula 3 in a suitable solvent such as methanol, ethanol, ortetrahydrofuran, and a base such as triethylamine (TEA), ordiisoproylethylamine (DIPEA), with heating to elevated temperature,e.g., 70° C. Further purification, resolution of stereoisomers,crystallization and/or preparation of salt forms may be carried outusing conventional techniques.

As will be apparent to those skilled in the art, in certain Instances,the starting or intermediate compounds in the synthesis may possessother functional groups which provide alternate reactive sites.Interference with such functional groups may be avoided by utilizationof appropriate protecting groups, such as amine or alcohol protectinggroups, and where applicable, appropriately prioritizing the syntheticsteps. Suitable protecting groups will be apparent to those skilled inthe art. Methods are well known in the art for installing and removingsuch protecting groups and such conventional techniques may be employedin the processes of the instant invention as well.

The following specific examples which are provided herein for purposesof Illustration only and do not limit the scope of the invention, whichis defined by the claims.

Material and methods. All reagent and solvents were purchased fromAldrich Chemical Corp. Chem-Impex International Inc. and TCI chemicalIndustry Co. Ltd. NMR spectra were obtained on either a Bruker AC 400(¹H NMR at 400 MHz and ¹³C NMR at 100 MHz) or a Bruker AC 300 (¹H NMR at300 MHz and ¹³C NMR at 75 MHz). Proton spectra were referenced totetramethylsilane as an internal standard and the carbon spectra werereferenced to CDCl₃, CD₃OD, or DMSO-d₆ (purchased from Aldrich orCambridge Isotope Laboratories, unless otherwise specified). Flashchromatography was performed on a Combiflash system (Combiflash Rf,Teledyne Isco) charged with silica gel column (Redi Sep. Rf, TeledyneIsco) or reverse phase column (High performance C18 Gold column). ESIMass spectra were obtained on a Shimadzu LCMS-2010 E V MassSpectrometer. HPLC analyses were obtained using a Waters XTerra MS C18 5μm 4.6×150 mm Analytical Column detected at 220 nm (unless otherwisespecified) on a Shimadzu Prominence HPLC system. The following timeprogram was used with a flow rate of 1.0 mL per minute:

Time Percent A Percent B (min) (H₂O with 0.05% TFA) (CH₃CN with 0.05%TFA)  2.50 90 10 20.00 10 90 30.00 10 90 32.50 90 10UPLC analyses were obtained using a Waters ACQUITY UPLC HSS T3 1.8 μm2.1×100 mm Analytical Column detected at 220 nm (unless otherwisespecified) on a Shimadzu Prominence UFLC system. The following timeprogram was used with a flow rate of 0.3 mL per minute:

Percent B Percent A (CH₃CN/Water 80:20% Time (H₂O with 0.05% NH₄COOHwith 0.05% NH₄COOH (min) and 0.1% HCOOH) and 0.1% HCOOH) 1.00 90 10 4.0030 70 5.00 30 70 5.50 90 10 6.50 90 10

Also provided herein (Scheme 2) is a method for preparation of compound(Ia),3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide,as defined herein before,

comprising the steps of:

-   -   (i) treating a compound of formula 14:

with a protected sugar,(4aR,6S,7R,8R,8aS)-2-phenylhexahydropyrano[3,2-d][1,3]dioxine-6,7,8-triol,of formula 15:

in the presence of a reducing agent, followed by a treatment of hexanalto form compound 16, benzyl4-(4-(2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)hexyl)amino)ethoxy)phenyl)butylcarbamate;

-   -   (ii) Subjecting compound 16 to catalytic hydrogenation to form        compound 17,        (1R,2S)-3-((2-(4-(4-aminobutyl)phenoxy)ethyl)hexyl)amino)-1-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propane-1,2-diol;        and

-   -   (iii) Condensing compound 17 with compound 2, methyl        3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate, in        the presence of base to form 19,        3,5-diamino-6-chloro-N—(N-(4-(4(2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4y)propyl)hexyl)amino)ethoxy)        phenyl) butyl)carbamimidoyl)pyrazine-2-carboxamide; and

-   -   (iv) hydrolyzing compound 19 in the presence of acid to form        (Ia).

An alternative process comprises replacing the compound of formula 16,above, with compound 27, followed by the hydrogenation, condensation,and hydrolysis steps just described to form compound (Ia).

Also provided herein (Scheme 3) is an alternate method for preparationof compound (Ia),3,5-diamino-6-chloro-N—(N-(4-(2-(2-hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide,as defined herein before.

EXAMPLES

The invention also comprises a compound prepared by the methods herein,or a pharmaceutically acceptable salt of the compound.

Synthesis of Ia,3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

Step 1

Preparation of benzyl4-(4(3-(tert-butyloxycarbonylamino)propoxy)phenyl)butyl carbamate(Compound 13)

To a solution of benzyl 4-(4-hydroxyphenyl)butyl carbamate (11, 60.0 g,300 mmol) in dry THF (600 mL) was added N-Boc ethanolamine (12, 38.7 g,300 mmol), Ph₃P (62.9 g, 300 mmol) and DIAD (48.6 g, 300 mmol) at 0° C.,then the reaction mixture was warmed to room temperature and stirredover night. The reaction mixture was concentrated in vacuum and theresidue was purified by column chromatography (silica gel, 15:85EA/hexanes) to afford desired compound 13 (50.0 g, 57%) as a yellowsolid: ¹H NMR (400 MHz, CDCl₃) δ 7.35 (m, 5H), 7.10 (d, J=8.0 Hz, 2H),6.80 (d, J=8.0 Hz, 2H), 5.10 (s, J=4.0 Hz, 2H), 4.0 (m, 2H), 3.5 (q,2H), 3.2 (q, 2H), 2.55 (t, J=8.0 Hz, 2H), 1.60 (m, 2H), 1.55 (m, 2H),1.45 (s, 9H).

Step 2

Preparation of Benzyl 4-(4-(2-aminoethoxy)phenyl)butylcarbamateHydrochloric Acid Salt (14)

Compound 13 (50.0 g, 112 mmol) was dissolved in 4 N HCl in dioxane (250mL) at room temperature and the solution was stirred for 1 hour. Afterconcentrated, the residue was suspended in MTBE (500 mL) and stirred for0.5 h. The solid is filtered out to afford hydrochloric acid salt 14(40.0 g, 83%) as a white solid: ¹H NMR (300 MHz, CD₃OD) δ 7.33 (m, 5H),7.10 (d, J=8.7 Hz, 2H), 6.88 (d, J=8.7 Hz, 2H), 5.05 (s, 2H), 4.18 (t,2H), 3.39 (m, 2H), 3.14 (t, J=7.2 Hz, 2H), 2.56 (t, J=7.5 Hz, 2H), 1.57(m, 4H).

Step 3

Preparation of Benzyl4-(4-(2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethoxy)phenyl)butylcarbamate(16)

A solution of hydrochloric acid salt 14 (13.5 g, 39.35 mmol) and triol15 (10.5 g, 39.35 mmol) in MeOH (150 mL) and AcOH (18.8 g, 314.8 mmol)was stirred at room temperature for 2 h, sodium cyanoborohydride (6.1 g,98.37 mmol) was added and the reaction mixture was stirred at roomtemperature overnight. Additional triol 15 (5.2 g, 19.67 mmol) was addedand the reaction mixture was stirred at room temperature for 4 h. Afterstarting material 14 was completely consumed, hexanal (5.9 g, 59.03mmol) was added and the reaction mixture was stirred at room temperaturefor 2 h. Solvent was removed in vacuum. The residue was washed with satdNa₂CO₃ (5.0 mL), azeotroped with MeOH and purified by columnchromatography (silica gel, 10:1 CH₂Cl₂/MeOH) to afford compound 16(12.2 g, 46% over two steps) as an off-white solid: ¹H NMR (300 MHz,CD₃OD) δ 7.45-7.44 (m, 3H), 7.31-7.29 (m, 9H), 7.05 (d, J=8.4 Hz, 2H),6.79 (d, J=8.4 Hz, 2H), 5.50 (s, 1H), 5.05 (s, 2H), 4.25-4.18 (m, 2H),4.03-3.87 (m, 6H), 3.78-3.55 (m, 3H), 3.13-2.96 (m, 6H), 2.85-2.69 (m,3H), 2.53 (t, J=6.7 Hz, 2H), 1.58-1.48 (m, 6H), 1.23 (br s, 6H), 0.86(t, J=6.1 Hz, 3H).

Step 4

Preparation of(1R,2S)-3-((2-(4-(4-aminobutyl)phenoxy)ethyl)(hexyl)amino)-1-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propane-1,2-diolAcetic Acid Salt (17)

A suspension of carbamate 16 (12.2 g, 17.99 mmol) and 10% Pd/C (3.66 g)in EtOH/AcOH (5:1, 120 mL) was subjected to hydrogenation conditions (1atm) overnight at room temperature. The reaction mixture was filteredthrough celite and washed with EtOH. The filtrate was concentrated invacuum to afford acetic salt 17 (9.40 g, 96%) as a colorless oil: ¹H NMR(300 MHz, CD₃OD) δ 7.48-7.44 (m, 2H), 7.32-7.30 (m, 3H), 7.11 (d, J=8.5Hz, 2H), 6.84 (d, J=8.5 Hz, 2H), 5.51 (s, 1H), 4.26-4.10 (m, 3H),3.95-3.91 (m, 2H), 3.78 (dd, J=1.8, 9.3 Hz, 1H), 3.60 (t. J=10.4, 1H),3.23-3.03 (m, 2H), 2.96-2.87 (m, 3H), 2.61-2.59 (m, 2H), 1.67-1.57 (m,6H), 1.31-1.25 (br s, 6H), 0.89 (t, J=6.6 Hz, 3H).

Step 5

Preparation of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-phenyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide (19)

To a solution acetic acid salt 7 (9.40 g, 17.27 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (18, 7.20 g, 27.64 mmol) in EtOH (75 mL) was added DIPEA (17.8g, 138.16 mmol) at room temperature. The reaction mixture was heated at70° C. in a sealed tube for 2 h, then cooled to room temperature, andconcentrated in vacuum. The residue was purified by columnchromatography (silica gel, 9:1 CH₂Cl₂/MeOH, 80:18:2 CHCl₃/MeOH/NH₄OH)to afford carboxamide 19 (9.20 g, 70%) as a yellow solid: ¹H NMR (300MHz, CD₃OD) δ 7.46-7.43 (m, 2H), 7.30-7.28 (m, 3H), 7.07 (d, J=8.6 Hz,2H), 6.79 (d, J=8.6 Hz, 2H), 5.48 (s, 1H), 4.22 (dd, J=3.9, 7.8 Hz, 1H),4.06-3.88 (m, 5H), 3.75 (dd, J=1.5, 6.9 Hz, 1H), 3.57 (t, J=10.5 Hz,1H), 3.25 (t, J=6.6 Hz, 2H), 2.93-2.83 (m, 3H), 2.68-2.56 (m, 5H),1.70-1.64 (m, 4H), 1.44-1.43 (m, 2H), 1.22 (m, 6H), 0.85 (t, J=8.1 Hz,3H).

Step 6

Preparation of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamideHydrochloric Acid Salt (Ia)

To a solution of carboxamide 19 (9.20 g, 12.16 mmol) in EtOH (30 mL) wasadded 4 N aq HCl (95 mL) at room temperature and the reaction mixturewas stirred for 4 h at room temperature. The reaction mixture wasconcentrated in vacuum and the residue was purified by reverse phasecolumn chromatography and lyophilized to afford hydrochloric acid saltIa (6.60 g, 81%) as a yellow hygroscopic solid: ¹H NMR (300 MHz, CD₃OD)δ 7.17 (d, J=8.4 Hz, 2H), 6.94 (d, J=8.4 Hz, 2H), 4.36 (br s, 2H),4.21-4.19 (m, 1H), 3.84-3.61 (m, 7H), 3.46-3.30 (m, 5H), 2.64 (t, J=6.5Hz, 2H), 1.80-1.69 (m, 6H), 1.36 (br s, 6H), 0.91 (t, J=6.6 Hz, 3H);ESI-MS m/z 669 [C₃₀H₄₉ClN₆O₇+H]⁺; Anal. (C₃₀H⁴⁹ClN₈O₇.2HCl.H₂O). Calcd.C, 47.40; H, 7.03; N, 14.74. Found, C, 47.11; H, 7.06; N, 14.54.

Alternate synthesis of I,3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

Step 1

Preparation of Benzyl4-(4-(3-((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-methyl-1,3-dioxan-4-yl)propylamino)propoxy)phenyl)butylcarbamate(26)

A solution of hydrochloric acid salt 14 (155 mg, 0.41 mmol) and triol 15(84 mg, 0.41 mmol) in MeOH (5.0 mL) was stirred at room temperature for0.5 h, then AcOH (0.036 mL, 0.6 mmol) and sodium cyanoborohydride (43mg, 0.6 mmol) was added and the reaction mixture was stirred at roomtemperature for 16 h. Solvent was removed in vacuum. The residue waswashed with satd Na₂CO₃ (5.0 mL), azeotroped with MeOH and purified bycolumn chromatography (silica gel, 10:1 CH₂Cl₂/MeOH, 10:1:0.1CHCl₃/MeOH/NH₄OH) to afford carbamate 26 (163 mg, 75%) as an white gummysolid: ¹H NMR (300 MHz, CD₃OD) δ 7.34-7.30 (m, 5H), 7.08-7.05 (m, 2H),6.85-6.82 (m, 2H), 5.06 (s, 2H), 4.70-4.67 (m, 1H), 4.08-3.96 (m, 4H),3.82-3.76 (m, 2H), 3.49-3.46 (m, 1H), 3.14-3.10 (m, 2H), 3.01-2.79 (m,4H), 2.65-2.45 (m, 2H), 2.05-2.01 (m, 2H), 1.59-1.49 (m, 4H), 1.27 (d,J=4.8 Hz, 3H).

Step 2

Preparation of Benzyl4-(4-(2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-methyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethoxy)phenyl)butylcarbamate(27)

A solution of carbamate 26 (1.02 g, 1.90 mmol), hexanal (380 mg, 3.80mmol), AcOH (0.33 mL, 5.70 mmol) and sodium cyanoborohydride (410 mg,5.70 mmol) in MeOH (30 mL) was stirred at room temperature for 16 h.Solvent was removed in vacuum. The residue was washed with satd Na₂CO₃(30 mL), azeotroped with MeOH and purified by column chromatography(silica gel, 10:1 CH₂Cl₂/MeOH) to afford carbamate 27 (990 mg, 84%) asan white gummy solid: ¹H NMR (300 MHz, CD₃OD) δ 7.35-7.31 (m, 5H), 7.06(d, J=8.4 Hz, 2H), 6.81 (d, J=8.4 Hz, 2H), 5.08 (s, 2H), 4.80 (br s,1H), 4.69-4.66 (m, 1H), 4.12 (dd, J=9.3, 2.4 Hz, 1H), 4.05-3.98 (m, 3H),3.84-3.76 (m, 2H), 3.54-3.48 (m, 1H), 3.38 (t, J=10.5 Hz, 1H), 3.20-2.96(m, 4H), 2.83 (d, J=6.0 Hz, 2H), 2.73-2.64 (m, 2H), 2.56 (t, J=7.2 Hz,2H), 1.63-1.50 (m, 6H), 1.32 (d, J=5.1 Hz, 3H), 1.27-1.24 (m, 6H), 0.87(d, J=6.6 Hz, 3H).

Step 3

Preparation of(R,2S)-3-((2-(4-(4-Aminobutyl)phenoxy)ethyl)(hexyl)amino)-1-((4R,5R)—S-hydroxy-2-methyl-1,3-dioxan-4-yl)propane-1,2-diolAcetic Acid Salt (28)

A suspension of carbamate 27 (890 mg, 1.44 mmol) and 10% Pd/C (400 mg)in MeOH/AcOH (5:1, 60 mL) was subjected to hydrogenation conditions (1atm) for 6 h at room temperature. The reaction mixture was filteredthrough celite and washed with MeOH. The filtrate was concentrated invacuum and crashed from ether to afford acetic salt 28 (782 mg, 90%) asa white gummy solid: ¹H NMR (300 MHz, CD₃OD) δ 7.09 (d, J=8.7 Hz, 2H),6.86 (d, J=8.7 Hz, 2H), 4.69-4.67 (m, 1H), 4.00-3.85 (m, 1H), 3.84-3.76(m, 2H), 3.53-3.51 (m, 1H), 3.38 (t, J=10.5 Hz, 1H), 2.98-2.59 (m, 10H),1.96 (s, 13H), 1.67-1.47 (m, 6H), 1.40-1.27 (m, 6H), 1.26 (d. J=5.1 Hz,3H), 0.88 (d, J=6.3 Hz, 3H).

Step 4

Preparation of3,5-Diamino-1-chloro-N—(N-(4-(4-(2-(((2S,3R)-2,3-dihydroxy-3-((4R,5R)-5-hydroxy-2-methyl-1,3-dioxan-4-yl)propyl)(hexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)Pyrazin-2-carboxamide (20)

To a solution acetic acid salt 28 (189 mg, 0.313 mmol) and methyl3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydroiodicacid salt (18, 192 mg, 0.502 mmol) in EtOH (8 mL) was added DIPEA (0.42mL, 2.50 mmol) at room temperature. The reaction mixture was heated at70° C. in a sealed tube for 2 h, then cooled to room temperature, andconcentrated in vacuum. The residue was purified by columnchromatography (silica gel, 9:1 CH₂Cl₂/MeOH, 80:18:2 CHCl₃/MeOH/NH₄OH)to afford carboxamide 20 (142 mg, 65%) as a yellow solid: ¹H NMR (300MHz, CD₃OD) δ 7.10 (d, J=8.1 Hz, 2H), 6.84 (d, J=8.1 Hz, 2H), 4.66 (q,J=5.1 Hz, 1H), 4.06-4.01 (m, 3H), 3.94-3.89 (m, 1H), 3.82-3.74 (m, 2H),3.49 (dd, J=9.3, 2.4 Hz, 1H), 2.96-2.78 (m, 3H), 2.67-2.61 (m, 5H),1.68-1.67 (m, 4H), 1.50-1.48 (m, 2H), 1.29 (br s, 6H), 1.25 (d, J=5.1Hz, 3H), 0.87 (t, J=6.9 Hz, 3H).

Preparation of3,5-Diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamideHydrochloric Acid Salt (Ia)

To a solution of carboxamide 20 (400 mg, 0.57 mmol) in EtOH (5 mL) wasadded 4 N aq HCl (15 mL) at room temperature and the reaction mixturewas heated at 55° C. for 24 h. After concentrated, the residue wasdissolved in 4 N aq HCl (15 mL) and heated at 65° C. for 16 h. Thereaction mixture was concentrated, crashed from EtOH/Et₂O, re-purifiedby preparative TLC and lyophilized to afford hydrochloric acid salt (Ia)(, 354 mg, 83%) as a yellow hygroscopic solid: ¹H NMR (300 MHz, D₂O) δ7.18 (d, J=8.1 Hz, 2H), 6.87 (d, J=8.1 Hz, 2H), 4.30 (br s, 2H),4.19-4.16 (m, 1H), 3.76-3.55 (m, 7H), 3.39-3.24 (m, 6H), 3.57 (t, J=5.4Hz, 2H), 1.65-1.64 (m, 6H), 1.30-1.19 (m, 6H), 0.78-0.75 (m, 3H); ESI-MSm/z 669 [C₃₀H₄₉ClN₆O₇+H]⁺.

Preparation of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide(Freebase of 1a)

3,5-diamino-6-chloro-N—(N-4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamidehydrochloric acid salt (12.51 g) was dissolved in 150 mL H₂O andtreated, stirring, with NaOH (0.1M aqueous, 435 mL) to give a gummyprecipitate. The liquid (pH ˜11) was decanted through a filter funnel(the majority of material adhered to the sides of the flask). Theresidue was treated with H₂O (2×300 mL), stirring anddecanting/filtering in a similar fashion. The remaining residue wassuspended in CH₃CN/H₂O/MeOH, and concentrated to provide a yellow-ambersolid, 9.55 g. ESI-MS m/z 669 [C₃₀H₄₉ClN₆O₇+H]⁺, purity 89% at 224 nm,90% at 272 nm, 82% at 304 nm, 63% by MS trace. The crude product washeated with isopropanol (100-150 mL) at 70° C. for 15 min, then filteredwarm. The solids were treated similarly with isopropanol twice more,heating for 30 minutes each time and allowing the mixture to cool (2hrs-O/N) before filtration. The resulting solids were dried to provide7.365 g of a yellow-amber amorphous solid, m.p. 133.1-135.6° C. (11.0mmol yield as free base). ¹H NMR (400 MHz, dmso) δ 9.31-7.34 (m, 4H),7.10 (d, J=8.6 Hz, 2H), 6.83 (d, J=8.5 Hz, 2H), 6.61 (br. s, 3H),4.76-4.09 (m, 5H), 3.98 (t, J=6.1 Hz, 2H), 3.71-3.62 (m, 2H), 3.59 (dd,J=10.8, 3.4 Hz, 1H), 3.54-3.46 (m, 1H), 3.43 (dd, J=7.9, 1.3 Hz, 1H),3.38 (dd, J=10.8, 5.9 Hz, 1H), 3.15 (br. s, 2H), 2.92-2.76 (m, 2H), 2.66(dd, J=13.1, 5.2 Hz, 1H), 2.58-2.51 (m, 4H), 2.46 (dd, J=13.1, 6.5 Hz,1H), 1.66-1.45 (m, 4H), 1.45-1.31 (m, 2H), 1.31-1.09 (m, 6H), 0.90-0.76(m, 3H). ¹³C NMR (101 MHz, dmso) δ 173.27, 160.96, 156.48, 154.68,151.14, 133.70, 129.05, 119.12, 117.53, 114.14, 72.23, 71.37, 70.60,70.04, 65.74, 63.34, 57.39, 54.72, 52.86, 40.10, 33.72, 31.15, 28.37,28.09, 26.38, 26.36, 22.02, 13.84. ESI-MS m/z 669 [C₃₀H₄₉ClN₈O₇+H]⁺.

Preparation of the 1-Hydroxy-2-naphthoate Salt of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

A mixture of 32.8 mg (0.049 mmol) of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl) carbamimidoyl)pyrazine-2-carboxamide, 164 μLof a 0.3 M solution of 1-hydroxy-2-naphthoic acid in methanol (0.049mmol of 1-hydroxy-2-naphthoic acid), and about 0.33 mL of methanol waswarmed on a hot plate set at 85° C. until all the solid dissolved. Thesolution was allowed to cool to ambient temperature. The solution wasplaced in a refrigerator (about 5° C.) and allowed to stand overnight,during which time crystallization occurred. The liquid was decanted andthe solid was dried in a stream of dry air to give 29.5 mg (62% yield)of the 1-hydroxy-2-naphthoate salt of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl) carbamimidoyl)pyrazine-2-carboxamide. ¹H NMR(500 MHz, DMSO) 8.2 (m, 1H), 7.7 (m, 2H), 7.4 (d, 1H), 7.3 (d, 1H), 7.1(m, 2H), 6.95 (m, 1H), 6.85 (m, 2H), 4.6-4.2 (m, 2H), 4.0 (m, 2H),3.8-3.6 (m, 2H), 3.6-3.2 (m, 6H), 2.9-2.5 (m, 6H), 1.6 (m, 4H), 1.4 (m,2H), 1.2 (m, 6H), 0.83 (m, 3H) ppm.

Preparation of the 1-Hydroxy-2-naphthoate Salt of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

A mixture of 105.3 mg (0.157 mmol) of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl) carbamimidoyl)pyrazine-2-carboxamide, 525 μLof a 0.3 M solution of 1-hydroxy-2-naphthoic acid in methanol (0.158mmol of 1-hydroxy-2-naphthoic acid), and about 1 mL of methanol waswarmed on a hot plate set at 85° C. until all the solid dissolved. Thesolution was allowed to cool to ambient temperature and placed in arefrigerator (about 5° C.). After about 20 min it became turbid and wasseeded. After about 2 hours solid was present A stir bar was added tothe mixture and it was stirred in the refrigerator overnight, duringwhich time it became very thick. Another 1.5 mL of methanol was addedand the slurry was stirred in the refrigerator overnight. The mixturewas centrifuged, the liquid was decanted, and the solid was dried in astream of dry air to give 74 mg (55% yield) of the1-hydroxy-2-naphthoate salt of3,5-diamino-6-chloro-N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide.

Pharmacology of Compound (Ia)3,5-diamino-6-chloro-N(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butylcarbamimidoyl)pyrazine-2-carboxamide

Assay 1. In Vitro Measure of Sodium Channel Blocking Activity andReversibility

One assay used to assess mechanism of action and/or potency of thecompounds of the present invention involves the determination of luminaldrug inhibition of airway epithelial sodium currents measured undershort circuit current (I_(SC)) using airway epithelial monolayersmounted in Ussing chambers. Cells are obtained from freshly excisedhuman, canine, sheep or rodent airways. This assay is described indetail in Hirsh, A. J., Zhang, J., Zamurs, A, et al. Pharmacologicalproperties ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidinemethanesulfonate (552-02), a novel epithelial sodium channel blockerwith potential clinical efficacy for CF lung disease. J. Pharmacol. Exp.Ther. 2008; 325(1): 77-88. Inhibition of transcellular sodium movementthrough ENaC was measured using polarized bronchial epithelial cellmonolayers mounted in a modified Ussing chamber. Primary cultures ofcanine or human bronchial epithelial cells grown using an air-liquidinterface were tested under voltage clamp conditions. The short-circuitcurrent (I_(SC)) was measured as an index of transepithelial sodiumtransport to assess potency.

Compound (Ia)3,5-diamino-6-chloro-N—(N-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamidewas a potent inhibitor of transcellular sodium transport and wasapproximately 60-fold more active than amiloride in canine bronchialepithelial cells (CBE), and approximately 160-fold in human bronchialepithelial cells (HBE) (FIG. 1). In CBE Compound (Ia) had an IC₅₀ of13.2±8.0 nM and in HBE Compound (Ia) had an IC₅ of 2.4±1.8 nM (Table 1).

TABLE 1 Inhibition of Short-Circut Current by Compound (Ia) in caninebronchial epithelial cells and human bronchial epithelial cells (IC₅₀nM) Compound I Species Amiloride (Parent) Canine 781.5 ± 331 (40) 13.2 ±8.0 (7)* Human 389 ± 188 (22) 2.4 ± 1.2 (4)* Values represent the mean ±SD (n) *Indicates significance (p < 0.05) from amiloride

Recovery of short circuit current (I_(SC)) from maximal block was usedas an indirect measurement of drug off-rate. Percent recovery of I_(SC)after full-block, determined after three apical surface washes andcalculated by the formula: recovered (I_(SC))/pre-treatment(I_(SC))×100, was significantly (22 fold) less reversible than amiloridein CBE and 9.5 fold less in HBE (Table 2), Indicating that Compound (Ia)produces a longer, more durable block on ENaC.

TABLE 2 Reversibility of Compound (Ia) on Short-Circuit Current inCanine Bronchial Epithelial Cells and Human Bronchial Epithelial Cells(% recovery) Species Amiloride Compound (1a) Canine 90.1 ± 27.6 (39) 4.1± 11.8 (7)* Human 89.5 ± 10.7 (4) 9.4 ± 17 (3)* Values represent themean ± SD (n) *Indicates significance (p < 0.05) from amilorideAssay 2. Mucociliary Clearance (MCC) Studies in Sheep

The animal model that has been used most often to measure changes in MCCis the sheep model. The effect of compounds for enhancing mucociliaryclearance (MCC) can be measured using an in vive model described bySabater et al., Journal of Applied Physiology, 1999, pp. 2191-2196,incorporated herein by reference.

In these studies, adult sheep were restrained and nasally intubated withan endotracheal tube. Aerosolized test articles were administered over10-15 minutes to sheep. Radiolabeled ^(99m)Tc-sulfur colloid (TSC, 3.1mg/mL; containing approximately 20 mCi) was then administered at aspecified time four or eight hours after test article. The radiolabeledaerosol was administered through the endotracheal tube for about 5minutes. The sheep were then extubated, and total radioactive counts inthe lung were measured every 5 minutes for a 1-hour observation period.The rate of radiolabel clearance from the lung is representative of theMCC rate in the animal. The advantage of this system is that it closelysimulates the human lung environment. The model also allows for thecollection of simultaneous PK/PD information through plasma and urinesampling over the test period. There are also several techniques tomeasure the drug concentrations on the airway surface during the MCCmeasurements. These include the collection of exhaled breath condensatesor a filter paper method to obtain ASL via bronchoscopy.

The ovine model described above was used to evaluate the in vivo effects(efficacy/durability) of aerosol-delivered Compound (Ia) on MCC.Treatments consisting of either 4 mL of Compound (Ia), ComparativeExample 1, Comparative Example 4, vehicle (sterile distilled H₂O), ortest agent in combination with HS were tested. To determine if combiningHS with Compound (Ia) MCC, HS was administered immediately followingCompound (Ia) administration. Test solutions were aerosolized using aRaindrop nebulizer at a flowrate of eight liters per minute andconnected to a dosimetry system consisting of a solenoid valve and asource of compressed air (20 psi). The deposited dose of drug in sheeplungs after an aerosol administration using the Raindrop nebulizer isestimated to be 8-15% of the dose. Using a Raindrop nebulizer,radiolabeled TSC was administered over approximately 3 minutes either 4or 8 hours after drug treatment to evaluate efficacy/durability.Radioactive counts were measured in a central region in the right lungat 5 min intervals for one hour with a gamma camera. Three methods ofanalysis were utilized, 1) initial rate of clearance (slope) over thefirst 30 min fitted using linear regression 2) area under the curve for% clearance over time over one hour, and 3) the maximum clearanceobtained in one hour.

The effect of Compound (Ia) at 16 μg/kg, 0.16 μg/kg and 0.016 μg/kg weretested and compared to vehicle (4 mL sterile H₂O) on sheep MCC four hourpost-dosing (FIG. 2). The analyses of effects are shown in Table 3. Atall doses tested, Compound (Ia) enhanced MCC compared to vehiclecontrol. The 16 μg/kg dose was considered to be a maximum MCC effect.

TABLE 3 MCC in Sheep at 4 h Post-dose of Compound (Ia) or VehicleCompound Initial Slope Maximum I Dose (4.0-4.5 h) AUC (% Cl - h)Clearance 16 μg/kg 39.0 ± 3.9* (4) 18.6 ± 2.2*^(†) (4) 33.8 ± 3.7*^(†)(4) 0.16 μg/kg 39.1 (2) 19 (2) 33.1 (2) 0.016 μg/kg 33.3 ± 4.4* (4) 14.4± 13* (4) 25.5 ± 1.3* (4) Vehicle 17.2 ± 6.8 (8) 7.3 ± 1.5 (8) 12.2 ±2.9 (8) (H₂O) 4 mL Data are reported as the mean ± SD (n) Study with n =2, not included in statistical analysis *Indicates significance (p <0.05 from vehicle. ^(†)Indicates significance (p < 0.05) from 0.016μg/kg dose.

To determine whether HS increases the MCC effect of Compound (Ia), HS(6.25 mL of 10% HS; 62.5 mg deposited, assuming 10% deposition) wasdosed Immediately following 0.016 μg/kg of Compound (Ia) and MCC wasassessed four hours after the combined dosing (FIG. 12). HS increasedthe effect of a 0.016 μg/kg dose of Compound (I) to a maximal effect, asseen with both the 0.16 and 16 μg/kg doses of Compound (Ia) alone (FIG.2). Therefore, a maximal MCC effect can be achieved when HS is added toa dose (0.016 μg/kg) of Compound (Ia) which produces a sub-maximalresponse when given without HS.

TABLE 4 MCC in Sheep at 4 h Post-dose of Vehicle, Compound (Ia) and HSInitial Slope Maximum Dose (4.0-4.5 h) AUC (% Cl h) Clearance Compound(Ia) 44.9 (2) 20.7 (2) 37.0 (2) (0.016 μg/kg; 4 mL + HS) Compound (Ia)33.3 ± 4.4 (4) 14.4 ± 1.3 (4) 25.5 ± 1.3 (4) (0.016 μg/kg; 4 mL) VehicleH₂O (4 mL) 17.2 ± 6.8 (8) 7.3 ± 1.5 (8) 12.2 ± 3 (8) Data are reportedas the mean ±_SD (n). Study with n = 2, not included in statisticalanalysis.

To assess both the durability of Compound (Ia) and the effect ofaddition of HS to Compound (Ia) MCC was measured eight hours post dosingwith vehicle (H₂O), HS 7% alone, 0.16, 1.6 and 16 μg/kg Compound (Ia)alone or a combination of 0.16 μg/kg Compound (Ia) and 7% HS (total 4 mlvolume for every treatment) (FIG. 4). After dosing with vehicle, the MCCat 4 and 8 hours was the same, indicating that the rate of MCC in thesheep over 4-8 hours is at a steady-state (FIGS. 12 and 13). Eight hoursfollowing 4 mL of 7% HS administration, no change in MCC was observedcompared to vehicle, indicating the HS effect had disappeared. All threedose groups (0.16, 1.6, and 16 μg/kg) of Compound (Ia) Increased MCC ina dose-related manner compared to both vehicle and HS, indicating thatCompound (Ia) has a longer duration of action than HS alone (FIG. 4).The combination dose of HS and Compound (Ia) increased the effect of a0.16 μg/kg dose of Compound (Ia) to greater than that observed for the16 μg/kg dose, indicating a 100 fold gain in activity when HS was addedto Compound (Ia) (FIG. 4). The enhancement of Compound (Ia) activity byHS, at a time when HS has no inherent activity, clearly indicatessynergy between HS and Compound (Ia).

TABLE 5 MCC in Sheep at 8 h Post-dose of Vehicle, HS, Compound (1a) or aCombination of HS and Compound (1a) Initial Slope Maximum Dose (8.0-8.5h) AUC (% Cl h) Clearance Vehicle - H₂O (4 mL) 17.8 ± 5.7 (4) 7.8 ± 1(4) 14.2 ± 0.7 (4) 7% HS (4 mL) 17.8 (2) 7.6 (2) 14.6 (2) Compound (Ia)24.0 (2) 10.7 (2) 19.7 (2) (0.16 μg/kg; 4 mL) Compound (Ia) 24.4 (2)11.1 (2) 21.2 (2) (1.6 μg/kg; 4 mL) Compound (Ia) 28.0 (2) 13.9 (2) 26.7(2) (16 μg/kg; 4 mL) Compound (Ia) 30.9 ± 2.5 (4) 15.3 ± 2.2 (4) 27.5 ±1.6 (4) (0.16 μg/kg + 7% HS; 4 mL) Data are reported as the mean ± SD(n). Study with n = 2, not included in statistical analysisAssay 3. d. Airway Surface Liquid Drug (ASL) Clearance and Metabolism byHuman Airway Epithelium

The disappearance of Compound (Ia) from the apical surface and airwayepithelial metabolism were assessed in HBE (Table 6). In theseexperiments 25 μL of a 25 μM solution of ENaC blocker was added to theapical surface of HBE cells grown at an air/liquid interface, and thedrug concentration in the apical and basolateral compartment wasmeasured over 2 h by UPLC. After 2 h incubation of Compound (Ia) on theapical surface (37° C.), no metabolites were detected on either theapical or basolateral sides and no Compound (Ia) was detectable on thebasolateral side.

TABLE 9 Apical Disappearance and Metabolism of Compound (1a) by HBE % on% of Initial Drug % of Apical % of Initial Basolateral Mass on ApicalMass as Apical Mass Side as Com- Side (Parent and Metabolites onBasolatoral Metabolites pound metabolite, 2 h) (2 h) Side (2 h) (2 h)Com- 80.7* ± 6.2% none none none pound (Ia)Assay 4. e. Airway Hydration and Sodium Channel Block (In Vitro Model)

Parion Sciences has developed experimental models for assessing airwayhydration in cell cultures (Hirsh, A. J., Sabater, J. R., Zamurs, A., etal. Evaluation of second generation amiloride analogs as therapy for CFlung disease. J. Pharmacol. Exp. Ther. 2004; 311(3): 929-38. Hirsh, A.J., Zhang, J., Zamurs. A., et al. Pharmacological properties ofN-(3,5-diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02), a novelepithelial sodium channel blocker with potential clinical efficacy forCF lung disease. J. Pharmacol. Exp. Ther. 2008; 325(1): 77-88).

Primary CBE cells are plated onto collagen-coated, porous membranesmaintained at an air-liquid interface to assess maintenance of surfaceliquid volume over time. At the start of the experiment, each 12 mmsnapwell insert was removed from the plate containing air-liquidinterface culture media, blotted dry, weighed, and 50 μL of vehicle(0.1% DMSO), or ENaC blocker (10 μM in 0.1% DMSO) applied to the apicalsurface and the mass was recorded. The inserts were immediately returnedto a transwell plate (500 μL, Krebs Ringer Bicarbonate (KRB), pH 7.4 inlower chamber) and placed in a 37° C., 5% CO₂ incubator. To reduceartifact due to an apical carbohydrate osmotic gradient upon water loss,glucose was not included in the apical buffer. Compound (Ia) was testedand compared to vehicle, and the mass of ASL was monitored serially from0-8 or 24 h. The mass of surface liquid was converted to volume in μL.Data are reported as % initial volume (100%=50 μL).

The duration of sodium transport inhibition was determined indirectly bymeasuring the buffer retained after a 50 μl volume of experimentalbuffer was added to the apical surface of CBE cells. Only 12.5±12.1% ofvehicle (buffer) remained on the surface after 8 hours and a smallincrease in surface liquid retention was seen with 10 μM amiloride inthe vehicle (25±19.2% after 8 hours). In comparison, Compound (Ia)significantly increased apical surface liquid retention, maintaining88.3±13% of the surface liquid over 8 hours (FIG. 5).

To test Compound (Ia) further, the duration of incubation was increasedfrom eight to 24 hours. Amiloride was not tested over 24 hours as themajority of the effect was gone after eight hours. After 24 hours, only11% of the vehicle buffer remained whereas, Compound (Ia) maintained72.3±7.3% of surface liquid over 24 hours, a loss of only 16% relativeto the 8-hour measure, suggesting Compound (Ia) exhibits a durableeffect on liquid retention (FIG. 6).

Comparative Examples

The present compound of formula (I) is more potent and/or absorbed lessrapidly from mucosal surfaces, especially airway surfaces, compared toknown sodium channel blockers, such as Comparative Examples 1 through 5,described below. Therefore, the compound of formula (I) has a longerhalf-life on mucosal surfaces compared to these compounds.

Comparative Examples 1 through 4 are claimed, described or within thedisclosures of WO 2003/070182 (U.S. Pat. Nos. 6,858,615; 7,186,833;7,189,719; 7,192,960; and 7,332,496), WO 2005/044180 (U.S. Appln. No.2005/0080093 and U.S. Pat. No. 7,745,442), WO 2004/073629 (U.S. Pat.Nos. 6,903,105; 6,995,160; 7,026,325; 7,030,117; 7,345,044; 7,820,078;and 7,875,619), WO 2005/016879 (U.S. Pat. Nos. 7,064,129; 7,247,637;7,317,013; 7,368,447; 7,368,451; 7,375,107; 7,388,013; 7,410,968; and7,868,010), or WO 2008/031028 (U.S. Patent Application Publications2008/0090841 and 2009/0082287) as sodium channel blockers having usefulmedicinal properties and can be prepared by methods described thereinand others known in the art.

The compound of Comparative Example 1 is included in the sodium channelblocking compounds of WO 2008/031028, where its structure can be seen onpage 14.

Comparative Example 2 is3,5-diamino-6-chloro-N—(N-(4-(4-(2-(dihexylamino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide,which is within the generic disclosure of WO 2004/073629.

Comparative Example 3 is3,5-diamino-6-chloro-N—(N-(4-(4-(2-(((2S,3R,4R,5R)-5-hydroxy-2,3,4,6-tetramethoxyhexyl)((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino) ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide, whichis within the generic disclosure of WO 2008/031028.

(S)-3,5-diamino-6-chloro-N—(N-(4-(4(2,3-diamino-3-oxopropoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamide

The compound of Comparative Example 4 can be seen on page 15 of US2005/0080093 and as Compound 2 on page 90 of WO 2008/031048, and asCompound 2 on pages 42-43 of WO 2008/031028. In order to have usefulactivity in treating Cystic Fibrosis and C.O.P.D a compound must haveproperties that will cause enhancement of mucociliary clearance (MCC) atdoses that do not elevate plasma potassium which will eventually lead tohyperkalemia, a serious and dangerous condition, on multiple dosing. Itmust therefore be avoided in this class of compounds, which are known toelevate plasma potassium if they are significantly excreted by thekidney. In order to evaluate this potential, it is beneficial to haveMCC activity in vivo and not cause elevation of plasma potassium at theuseful dose. One model to assess this is the sheep MCC model describedbelow. As can be seen from the Table 7 below, the ED₅₀ (AUC=47%) forComparative Example 1 in the sheep MCC model is approximately 3000 μM.

TABLE 7 Change from Vehicle in MCC Effect at 8 hrs. in Sheep Using 3Different Measures Max Slope AUC Clearance Approximate Table 1. (8-8.5h) (% Cl*h) (%) ED₅₀ 300 μM (Ia) 10.2 (100%) 6.2 (100%) 12.8 (100%) 30μM (Ia) 6.6 (65%) 3.3 (53%) 7.0 (55%) 2.4 nmol/kg 3000 μM 6.1 (60%) 2.9(47%) 4 (31%) 240 nmol/kg (Comp. Ex. 1) Maximal 10.2 (100%) 6.2 (100%)12.8 (100%) Effect

As can be seen from the Table 7 and FIG. 7 the ED₅₀ for ComparativeExample 1 in the sheep MCC model is approximately 240 nmol/kg (3 mM)using three different measures (slope, AUC and Maximum Clearance). Atthis dose, which would be a clinically active dose, Comparative Example1 causes a rise in plasma potassium which on repeat dose will lead tohyperkalemia (FIG. 8). Thus, Comparative Example I is unacceptable forhuman use while Compound (Ia) produces a safe and effective MCC with abenefit to risk ratio greater than 1000 in this model.

In order to lower the potential renal effect of the molecule, morelipophilic compounds were examined. Comparative Example 2 which replacesthe two hydrophilic groups of Comparative Example 1 with two lipophilicchains of equal length results in compound Comparative Example 2 whichis an order of magnitude less potent than Comparative Example 1 in vitro(Table 8) and thus unsuitable to produce sustained MCC in vivo.Comparative Example 3 in which all of the oxygen's of ComparativeExample 1 were retained and 5 of the hydroxyl's methyl groups were addedto 5 of the hydroxyl's had a similar decrement in in vitro activity. Itthus appeared that it was not possible to produce an active and renallysafe molecule from this structural frame work. It was unexpectedtherefore that compound (Ia) was discovered to retain in vitro activityequal to Comparative Example 1. Even more surprising and unexpected wasthat Compound (Ia) was over 100 times more potent in vivo thanComparative Example I and caused no rise in plasma potassium ateffective MCC doses.

TABLE 8 In Vitro Measure of Sodium Channel Blocking Activity CompoundIC₅₀ (nM) Ia 13.2 Comparative 11.8 Example 1 Comparative 124.5 Example 2Comparative 144.1 Example 3 Comparative 6.6 Example 4

Another compound which has been studied extensively is the compound ofComparative Example 4,(S)-3,5-diamino-6-chloro-N—N-(4-(4-(2,3-diamino-3-oxopropoxy)phenyl)butyl) carbamimidoyl)pyrazine-2-carboxamide.

The disappearance of Compound (Ia) from the apical surface and airwayepithelial metabolism were assessed in HBE and compared to ComparativeExample 4 (Table 9). In these experiments 25 μL of a 25 μM solution ofENaC blocker was added to the apical surface of HBE cells grown at anair/liquid interface, and the drug concentration in the apical andbasolateral compartment was measured over 2 h by UPLC. After 2 hincubation of Compound (Ia) on the apical surface (37° C.), nometabolites were detected on either the apical or basolateral sides andno Compound (Ia) was detectable on the basolateral side. In contrast,most of Comparative Example 4 was eliminated from the apical side with83% metabolized to the less active carboxylic acid,(S)-2-amino-3-(4-(4-(3-(3,5-diamino-6-chloropyrazine-2-carbonyl)guanidino)butyl)phenoxy)propanoic acid, structure below.

TABLE 9 Apical Disappearance and Metabolism of Compound (Ia) by HBE % ofInitial % of Initial % on Baso- Drug Mass on % of Apical Apical Masslateral Apical Side Mass as on Side as (Parent and MetabolitesBasolateral Metabolites Compound metabolite, 2 h) (2 h) Side (2 h) (2 h)Compound 80.7* ± 6.2% none none none (Ia) Comparative  41.6 ± 7.6% 83.0± 3.5% 8.3 ± 0.2 94.7 ± 1.0% Example 4 Values represent the mean ± SD*Indicates significantly different (p < 0.05) from Comparative Example4.

Compound (Ia) is 10,000 times more potent in sheep MCC than ComparativeExample 4 with no elevation of Plasma K, whereas Comparative Example 4has elevations of plasma K at the approximate ED50 dose of 3 mM (FIGS. 9and 10). This, again, demonstrates the unique unexpected potency andsafety advantage of compound Ia.

TABLE 10 MCC in Sheep at 4 h Post-dose of vehicle, Comparative Example 4or Compound (1a) Initial Slope Maximum Dose (4.0-4.5 h) AUC (% Cl × h)Clearance Comparative 32.2 ± 7.3* (6) 14.1 ± 2.2* (6) 22.9 ± 2.1* (6)Example 4 (112 μg/kg; 4 mL) Comparative 14.5 ± 1.3 (3) 6.9 ± 1.0 (3)14.6 ± 0.9 (3) Example 4 (11.2 μg/kg; 4 mL) Compound (Ia) 33.3 ± 4.4*(4) 14.4 ± 1.3* (4) 25.5 ± 1.3* (4) (0.016 μg/kg; 4 mL) Vehicle H₂O 17.2± 6.8 (8) 7.3 ± 1.5 (8) 12.2 ± 2.9 (8) (4 mL)

It has now been shown that the enhanced renal safety of Compound (Ia)can be explained by the marked reduction in clearance of drug by thekidney. If the compound can be kept away from sodium channels in thekidney, hyperkalemia should be markedly reduced. Following intravenousadministration to sheep, 43% of Comparative Example 1 was recovered inurine, whereas only 5% of compound I was recovered from urine. Even moredramatic is the surprising reduction in urinary recovery of drug whenadministered as an aerosol, directly into the lung. When ComparativeExample 4 is administered to sheep as an inhalation aerosol, 7.% of thedose is recovered in urine whereas only 0.07% of an aerosolized dose ofCompound (Ia) is recovered in urine. Reduced clearance of compound intourine (10-100-fold), combined with the above-described significantreduction in dose requirement leads to an unexpected 100,000 to1,000,000-fold difference in risk:benefit.

TABLE 11 Urine Excretion of Compound (Ia) and Comparative Example 4 inSheep. Assay Comp. Ex. 4 Compound (Ia) Log D 0.64 2.2 IC50 6.6 ± 3.7 nM13 ± 8 nM Human Plasma t½ = 37 min None Metabolism Human Plasma 76 ±2%    97 ± 2%  Protein Binding Urinary excretion of 7% 0.07%administered dose (sheep)

FIG. 9 graphs the percentage mucus clearance over time by Compound (Ia),3,5-Diamino-6-chloro-N—(N—(N-(4-(4-(2-(hexyl((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexyl)amino)ethoxy)phenyl)butyl)carbamimidoyl)pyrazine-2-carboxamidehydrochloric acid salt, and Comparative Example 4, as described in theMCC model, above. A similar percentage mucus clearance was provided byCompound (Ia) at a 7,000-fold lower dose than seen with ComparativeExample 4. Compound (Ia) provided a maximal effect in a clinicallyrelevant dose range.

FIG. 10 illustrates the significant increase in plasma potassium levelsat an efficacious dose seen in the plasma of the sheep receivingComparative Example 4 in the MCC study, above, over time. No effect inplasma potassium levels was seen at any dose tested in the sheepreceiving Compound (Ia).

That which is claimed is:
 1. A pharmaceutical composition comprising acompound of Formula (I):

or a pharmaceutically acceptable salt thereof, and an osmolyte.
 2. Thepharmaceutical composition of claim 1, wherein the osmolyte ishypertonic saline.
 3. The pharmaceutical composition of 2, wherein thehypertonic saline has a concentration of 4%-5% w/v.
 4. Thepharmaceutical composition of claim 2, wherein the hypertonic saline hasa concentration of about 4% w/v.
 5. The pharmaceutical composition ofclaim 1, wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 6. The pharmaceuticalcomposition of claim 1, wherein the compound is of the formula:

or a pharmaceutically acceptable salt thereof.
 7. The pharmaceuticalcomposition according to claim 1, wherein said composition is suitablefor inhalation.
 8. The pharmaceutical composition according to claim 1,wherein said composition is a solution for aerosolization andadministration by nebulizer.
 9. The pharmaceutical composition accordingto claim 1, wherein said composition is suitable for administration bymetered dose inhaler.
 10. The pharmaceutical composition according toclaim 1, wherein said composition is a dry powder suitable foradministration by dry powder inhaler.
 11. A pharmaceutical compositioncomprising a compound of the following formula:

or a pharmaceutically acceptable salt thereof, and hypertonic saline.12. The pharmaceutical composition of 11, wherein the hypertonic salinehas a concentration of 4%-5% w/v.
 13. The pharmaceutical composition ofclaim 11, wherein the hypertonic saline has a concentration of about 4%w/v.
 14. The pharmaceutical composition according to claim 11, whereinsaid composition is suitable for inhalation.
 15. The pharmaceuticalcomposition according to claim 11, wherein said composition is asolution for aerosolization and administration by nebulizer.
 16. Thepharmaceutical composition according to claim 11, wherein saidcomposition is suitable for administration by metered dose inhaler.